In Vitro Plasmid DNA Cleavage by Chromium(V ... - ACS Publications

Mar 25, 1999 - substantial DNA cleavage at pH 4.0-8.0 [[Cr(V)]0 ) 0.010-0.75 mM, phosphate buffer, and. 37 °C]. ... induce strand breaks in plasmid D...
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Chem. Res. Toxicol. 1999, 12, 371-381

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In Vitro Plasmid DNA Cleavage by Chromium(V) and -(IV) 2-Hydroxycarboxylato Complexes Aviva Levina,† Gregory Barr-David,† Rachel Codd,† Peter A. Lay,*,† Nicholas E. Dixon,‡ Anders Hammershøi,‡,§ and Philip Hendry‡,| School of Chemistry, University of Sydney, Sydney 2006 NSW, Australia, and Research School of Chemistry, Australian National University, Canberra 0200 ACT, Australia Received October 12, 1998

The ability of relatively stable Cr(V) and Cr(IV) complexes with 2-hydroxycarboxylato ligands [2-ethyl-2-hydroxybutanoate(2-) ) ehba; (1R,3R,4R,5R)-1,3,4,5-tetrahydroxycyclohexanecarboxylate(2-) ) quinate ) qa] to induce single-strand breaks in plasmid DNA has been studied under a wide range of reaction conditions. The Cr(V) complex, Na[CrVO(ehba)2], causes substantial DNA cleavage at pH 4.0-8.0 [[Cr(V)]0 ) 0.010-0.75 mM, phosphate buffer, and 37 °C]. The DNA cleavage is inhibited by the presence of excess ligand, by exclusion of O2, or by addition of organic compounds, such as alcohols, carboxylic acids, or DMSO, but it is not affected by traces of catalytic metals [Fe(III) or Cu(II)] or by addition of catalase. The Cr(IV)qa complexes, unlike the Cr(V) complexes, are able to cleave DNA in the presence of the ligand in a large excess [[Cr(IV)]0 ) 0.50 mM, [qa] ) 20-100 mM, pH 3.5-6.0, and 37 °C]. This is the first direct evidence for DNA cleavage induced by well-characterized Cr(IV) complexes. The proposed mechanism for DNA cleavage includes the following: (i) partial aquation of the bischelated Cr(V) and -(IV) complexes with the formation of reactive monochelated forms, (ii) binding of the Cr(V) and -(IV) monochelates to the phosphate backbone of DNA, (iii) one- or two-electron oxidations at the deoxyribose moieties of DNA by Cr(V) and -(IV), and (iv) cleavage of the resulting DNA radicals or cations with or without participation of O2. The patterns of DNA damage by Cr(V) and -(IV) can include strand breaks, generation of abasic sites, and the formation of Cr(III)-DNA complexes.

Introduction Chromium(VI) compounds are established carcinogens (1, and references therein). However, appreciable DNA damage by Cr(VI) is observed only in the presence of reductants (2, 3, and references therein). Wetterhahn and co-workers (4) suggested that the reason for such damage is the formation of reactive Cr(V) and -(IV) intermediates during the reduction of Cr(VI) to Cr(III). The ability of a relatively stable (5, 6) and well-characterized (7, and references therein) Cr(V) complex, Na[CrVO(ehba)2], to induce strand breaks in plasmid DNA at pH 3.8-4.8 was first demonstrated by Lay and co-workers (8). In subsequent publications, the ability of Na[CrVO(ehba)2] to cause oxidative cleavage (at pH 5.0-7.5) in doublestranded (9) and single-stranded (9,10)1 calf thymus (CT)2 DNA, as well as in thymidine nucleotides (11), has been †

University of Sydney. Australian National University. On leave from Department of Chemistry, University of Copenhagen, DK-2100 Copenhagen, Denmark. | Current address: CSIRO Molecular Science, P.O. Box 184, North Ryde, 1670 New South Wales, Australia. 1 In the work of Bose et al. (10), the reactions of CT DNA with Na[CrVO(ehba)2] were carried out in bis-Tris buffer, which forms relatively stable polyol complexes with Cr(V). 2 Abbreviations: bis-Tris, bis(hydroxyethyl)amino tris(hydroxymethyl)methane; hmba, 2-hydroxy-2-methylbutanoate(2-); ehba, 2-ethyl2-hydroxybutanoate(2-); qa or quinate, (1R,3R,4R,5R)-1,3,4,5-tetrahydroxycyclohexanecarboxylate(2-); CT, calf thymus; DTPA, diethylenetriaminepentaacetic acid; DMSO, dimethyl sulfoxide; EDTA, ethylenediamine-N,N,N′,N′-tetraacetic acid; GSH, glutathione (γ-Glu-Cys-Gly); HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; SSB, singlestrand break. ‡ §

reported. A number of Cr(V) 2-hydroxycarboxylato complexes, including Na[CrVO(hmba)2] and K[CrVO(qa)2], have been shown to promote cleavage of plasmid DNA in slightly acidic aqueous solutions (pH 3.8) (12). However, no studies have been reported on the interactions of plasmid DNA with Cr(V) 2-hydroxycarboxylato complexes in neutral media. Studies of DNA interactions with Cr(IV) complexes are more difficult than those with Cr(V) because (i) Cr(IV) complexes are generally less stable in aqueous media than the corresponding Cr(V) complexes (13) and (ii) EPR spectroscopy, which is a very powerful tool in Cr(V) chemistry, is much less applicable for Cr(IV) complexes (7, 14, and references therein). In most systems, no technique is available for direct observation of Cr(IV) intermediates formed during the Cr(VI) and -(V) reductions. Therefore, several indirect methods have been used to show potential roles of Cr(IV) complexes in Cr(VI)- and -(V)-induced genotoxicities. Lay and co-workers (15) were the first to suggest that transient Cr(IV)-ehba complexes, formed in the reaction of Na[CrVO(ehba)2] with V(IV), are more powerful DNA cleaving agents than the parent Cr(V) complex. Inhibition of DNA cleavage by Mn(II), which was believed to be a selective trap for Cr(IV), was proposed as an indicator of the roles of Cr(IV) in DNA damage by the Cr(VI) and reductant systems (16, 17). However, recent studies (18) have shown that Mn(II) is able to inhibit DNA cleavage in these systems by several ways, including reduction of Cr(V) complexes and of active oxygen species. Shi and co-workers (19, 20) used

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isolated products of Cr(VI) reductions, believed to be Cr(IV) complexes, for DNA cleavage studies. However, no unambiguous evidence for the existence and stability of Cr(IV) under the conditions of DNA cleavage was presented (19, 20). Recently, convenient methods for the quantitative in situ generation of Cr(IV) complexes with tertiary 2-hydroxycarboxylic acids, such as ehbaH2 or qaH2, have been found (21, 22), and the aqueous chemistry of these Cr(IV) complexes has been studied in detail (22-25). The concentrations of these Cr(IV) complexes in solutions can be readily determined from their strong absorbances in the visible region (22). Thus, direct studies of DNA interactions with well-characterized Cr(IV) complexes are now possible.

The 2-hydroxycarboxylato ligands, such as ehba and qa, are good models for intracellular 2-hydroxycarboxylates such as citrate and lactate. Chromium(V) and -(IV) 2-hydroxycarboxylato complexes are likely to be formed in vivo at pH 4.5-5.5, i.e., under the conditions of uptake and solubilization of insoluble chromates by phagocytosis (12, 14, 22). On the other hand, the 1,2-diolato moieties of the multifunctional ligand, qa, mimic those of D-glucose and other carbohydrates. Complexation with carbohydrates is the most likely method of stabilization of Cr(V) in vivo at pH g7 (12, 14, 26). The current work was aimed toward (i) a detailed study of plasmid DNA cleavage induced by Na[CrVO(ehba)2] (8), using a wide range of reaction conditions; (ii) a comparison of the DNA cleaving abilities of Cr(V) and Cr(IV) 2-hydroxycarboxylato complexes; and (iii) determination of the likely mechanisms of DNA interactions with Cr(V) and -(IV) complexes.

Experimental Procedures Caution: As(III) and Cr(VI) compounds are human carcinogens (1, 27), and Cr(V) and Cr(IV) complexes are mutagenic and potentially carcinogenic (8, 15). Contact with skin and inhalation must be avoided. Reagents. The following commercial reagents of analytical grade were used without further purification: As2O3, As2O5, H3BO3, CD3COOD, butanoic acid, 1-butanol, tert-butyl alcohol, diethylenetriaminepentaacetic actd (DTPA), 2-ethyl-2-hydroxybutanoic acid (ehbaH2), dimethyl sulfoxide (DMSO), and DMSOd6 (all from Aldrich, Milwaukee, WI); DNA grade agarose, Tris, and Chelex 100 (all from Bio-Rad, Hercules, CA); quinic acid (qaH2) (from ICN Biomedicals, Aurora, OH); L-ascorbic acid, bromophenol blue, catalase (EC 1.11.1.6, 2900 units/mg of protein), ethidium bromide, the reduced form of glutathione (GSH),4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid(HEPES) hemisodium salt, and sucrose (all from Sigma, St. Louis, MO); and NaH2PO4‚H2O, CH3COONa, Na2CrO4‚4H2O, H2O2 (30% aqueous solution), NaOH, HClO4, FeCl3‚6H2O, CuSO4‚5H2O, and ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA) disodium salt (all from Merck, Darmstadt, Germany). Water was purified using a Milli-Q system. The Cr(V) complexes, Na[CrVO(ehba)2] and K[CrVO(qa)2], were synthesized by known methods (5, 12), and their purities were confirmed by UV/vis and EPR spectroscopy. Plasmid pUC9 DNA (2.67 × 103 base pairs) was

Levina et al. prepared from an Escherichia coli strain and dialyzed into water as described previously (8). DNA Cleavage Assays. The ability of Cr(V) and -(IV) complexes to induce DNA cleavage was determined from the decrease in the relative concentrations of form I (covalently closed supercoiled) DNA and increases in those of form II (nicked circular) DNA and form III (linear) DNA (8). Stock solutions of 1.0 M NaH2PO4, CH3COONa, ehbaH2, or qaH2, used for preparation of the reaction buffers, were stirred overnight with ∼10 g/L Chelex 100 chelating resin, then filtered, and kept at 4 °C. The reaction buffers were prepared daily and assessed for traces of catalytic metals using Buettner’s ascorbate method (28). The concentration of the catalytic metal ions in the buffers corresponded to 6, linkage isomers are formed by Cr(V) binding to the 3,4,5-OH groups of qa (12).

Plasmid DNA Cleavage by Chromium(V) and -(IV)

Figure 11. Influence of qa concentration on DNA cleavage induced by Cr(V)- and -(V)-qa complexes. Conditions: [DNA] ) 19 mg/L, [Cr(IV)]0 or [Cr(V)]0 ) 0.50 mM, pH 4.0, [phosphate] ) 100 mM, reaction time of 1 h, and 37 °C.

with the Cr(IV)-qa complexes were observed at qa concentrations of 10-20 mM; a further increase of the qa concentration (up to 100 mM) caused a significant inhibition of DNA cleavage (Figure 11, at pH 4.0, and Figure S3 in the Supporting Information, at pH 5.0). However, this inhibition by excess qa ligand was less pronounced for the Cr(IV)-qa complexes than for the Cr(V)-qa complexes [Figures 11 and S3 (Supporting Information)].

Discussion Evidence for Direct Interactions of Cr(V) and -(IV) Complexes with DNA. Extensive discussions have been devoted to the possible natures of active species in Cr(VI)-induced genotoxicities (2, 3). The following two alternatives are considered. (i) DNA is damaged by direct oxidation by the highly reactive Cr(V) and -(IV) species formed during the reactions of Cr(VI) with intracellular reductants (direct pathway) (35), and (ii) intracellular reactions of Cr(VI), -(V), and -(IV) with H2O2 and/or O2 in the presence of reductants lead to the formation of •OH and other radical species capable of damaging DNA (indirect pathway) (36). The published evidence (8-11) for oxidation of DNA and nucleotides by the relatively stable Cr(V) complex, Na[CrVO(ehba)2], strongly suggests that direct reactions of DNA with Cr(V) and -(IV) are at least partially responsible for Cr(VI)-induced genotoxicities. This suggestion is reinforced by the results of the work described here. Although Na[CrVO(ehba)2] undergoes fast disproportionation in neutral aqueous solutions in the absence of excess ligand (6),4 its solutions are able to cleave DNA as long as a significant amount of Cr(V) is present (Figures 2 and 3). Indirect DNA oxidation mediated by radical species such as •OH is ruled out as (i) the reaction solutions do not contain H2O2 or strong reductants; (ii) in contrast to typical DNA cleavage reactions involving active oxygen species (35-37), the reaction of Cr(V)-ehba with DNA is not catalyzed by Fe(III) or Cu(II) and is not inhibited by catalase; and (iii) the reactions of Cr(V)-ehba with DNA (acetate or phosphate buffers, at pH 3.8 or 7.0) do not lead to a significant level of O2 consumption (25). The latter does not contradict the observed O2 dependencies of the DNA cleavage (Figure 5). Indeed, as the yields of oxidized DNA products are extremely low (see Experimental Procedures), the amounts of O2 consumed during the DNA

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oxidation are below the limits of detection. The proposed mechanism of plasmid DNA cleavage by Cr(V) and -(IV) 2-hydroxycarboxylato complexes is presented in Schemes 1 and 2. Role of Monochelated Cr(V) and -(IV) 2-Hydroxycarboxylato Complexes in DNA Cleavage. Inhibition of DNA cleavage by excess ligand, observed with both Cr(V) and Cr(IV) complexes [Figures 9, 11, and S3 (Supporting Information)], suggests that the initial bischelated forms of the complexes (Ia and IIa in Scheme 1) undergo partial aquation to form the reactive monochelated forms Ib and IIb (7, 8, 22) (eqs 2 and 3 in Scheme 1). Inhibition of DNA cleavage by excess ehba at pH 7.0 is partially due to H-donor properties of this compound, similar to those of other alcohols and carboxylic acids (Figure 8). However, this is not the only effect contributing to the inhibition by ehba. Indeed, unlike for all other inhibitors used, the inhibition by ehba is more pronounced at the lower pH value (Figure 9). Experimental evidence for the existence of monochelated Cr(V) and -(IV) oxo complexes has been presented (7, 22, 38, 39), but direct studies of such species are difficult due to their high reactivities. The exact structures of Cr(V) and -(IV) monochelates are yet to be established. Six-coordinate, octahedral structures proposed for reactive species Ib and IIb (Scheme 1) may explain why they are stronger oxidants in comparison with parent complexes Ia and IIa [which have distorted trigonal bipyramidal structures (22, 40)], as the high kinetic barrier en route to octahedral (13) Cr(III) complexes (24) is eliminated for Ib and IIb. Formation of octahedral complexes simply by addition of a sixth ligand to Ia or IIa is unlikely due to the steric demands (24). In addition, the loss of one 2-hydroxycarboxylato ligand in Ia and IIa would remove the negative charge of these complexes (eqs 2 and 3 in Scheme 1). The resulting uncharged species Ib and IIb would be expected to be more reactive toward DNA due to the absence of electrostatic repulsion from the negatively charged DNA backbone. The propensity of Cr(IV) 2-hydroxycarboxylato complexes IIa to lose one ligand to form IIb is much higher than for the corresponding Cr(V) complexes (22). This is the reason for the lower sensitivity to the presence of excess ligand of DNA cleavage by the Cr(IV) complexes [Figures 11 and S3 (Supporting Information)]. The reactive species Ib or IIb is likely to bind to the phosphate backbone of DNA molecule 1 (eqs 4 and 6 in Scheme 1) (8). This suggestion is supported by the EPR spectroscopic evidence for the binding of the Cr(V)-ehba complexes to inorganic phosphate (41), as well as to the phosphate groups of single-stranded DNA (10). The phosphate groups have been also shown to be responsible for binding of Cr(III) complexes to DNA (42). In the resulting Cr(V)- and -(IV)-DNA complexes 2 and 5, H bonds are likely to exist between the oxo ligands and the H atoms of the deoxyribose ring, thus facilitating the following redox reactions (eqs 5 and 6 in Scheme 1). Possible Mechanisms of Electron Transfer in Cr(V)- and -(IV)-DNA Complexes. As for most of the known DNA reactions with the complexes of transition metals at higher oxidation states [including Mn(III) porphyrins (37), Fe(V) and -(IV) oxo complexes in the activated form of iron bleomycin (37), and Ru(IV) oxo complexes (43)], oxidative DNA cleavage by Cr(V) and -(IV) oxo complexes I and II is likely to occur through H abstraction from the deoxyribose moiety (9-11). Sugden and Wetterhahn (9, 11) have found, by studies of cleavage

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Levina et al. Scheme 1

products, that C4′ atoms are the main targets in the oxidations of CT DNA and nucleotides by Na[CrVO(ehba)2] (acetate or phosphate buffers, at pH 5.0-7.5). On the other hand, Bose and co-workers (10) found that the oxidative cleavage of DNA occurs mainly at the C1′ position in the system Na[CrVO(ehba)2]/bis-Tris buffer, where Cr(V)-bis-Tris complexes are the main reacting species. Conditions of DNA cleavage in the work of Sugden and Wetterhahn (for double-stranded DNA in the absence of H2O2) (9) resemble those used in this work, except for the higher DNA concentratins and initial Cr(V) concentrations. Yields of DNA cleavage products (∼0.05% of the DNA concentration) are similar in both works, which points to similar cleavage mechanisms (given that the reaction is of first-order with respect to DNA). For the reactions of Cr(V) and -(IV) with pUC9 DNA under the conditions of the work described here, studies of the DNA cleavage products are difficult due to the very low concentrations of the oxidized DNA fragments. However, the results [Figures 1-11 and S1-S3 (Supporting Information)] are consistent with the assumption that the Cr(V) and -(IV) 2-hydroxycarboxylato complexes oxidize plasmid DNA mainly at C4′ positions (Scheme 1).

The redox reactions within Cr(V)-DNA complex 2 (eq 5 in Scheme 1) may occur through a one-electron pathway leading to DNA radical 3. Cr(IV) complex IIb is kinetically labile (24) and may dissociate from DNA in a fast stage, whereas 3 is stable for several milliseconds in the absence of O2 (44). Alternatively, 2 could react through a two-electron pathway with the formation of a DNA carbocation 4 [dissociation of the kinetically inert (13) Cr(III) complex from 4 is likely to be slow on the time scale of the redox reaction]. One-electron reduction of Cr(IV)-DNA complex 5 is likely to lead to DNA radical 6, retaining the attached inert Cr(III) complex (eq 6 in Scheme 1). O2-Dependent and O2-Independent Pathways for DNA Cleavage. Scheme 2 depicts the possible pathways for the further conversions of the intermediates formed in DNA reactions with Cr(V) and -(IV). DNA radicals 3 and 6 are likely to react in similar pathways, so only the reactions of the latter are shown (eqs 7-9 in Scheme 2). The O2-dependent reactions of such radicals (eq 7 in Scheme 2) are well-characterized (37, 45) and lead to peroxo radical 7, which is converted after a series of rearrangements to 3′-phosphoglycolate 8 and base pro-

Plasmid DNA Cleavage by Chromium(V) and -(IV)

Chem. Res. Toxicol., Vol. 12, No. 4, 1999 379 Scheme 2

penal 9, leaving the Cr(III) complex of 5′-phosphorylated end 10. This pathway is responsible for most of the plasmid DNA cleavage events under aerobic conditions at pH 7.0 (Figure 5). It also corresponds to the O2dependent formation of base propenals in the reactions of Cr(V) with CT DNA (9). 4′-DNA radical 6 is able to dissociate spontaneously in the absence of O2 (44) with the formation of cation radical 11 and Cr(III) complex 12 (eq 8 in Scheme 2). This reaction, which is likely to be facilitated by a decrease in the pH values, may be responsible for the significant anaerobic DNA cleavage at pH 4.0 (Figure 5). Carbocation 4, formed in the two-electron oxidation of DNA, is likely to be converted, after a release of free base (37), to the Cr(III) complex of oxidized DNA 14 (eq 10 in Scheme 2). The release of free bases in the O2-independent pathway has been observed (9) during the reactions of CT DNA with Cr(V). Notably, the detected amounts of free bases were higher than the amounts of the products arising from the oxidation of deoxyribose rings (9). This is consistent with the release of free base leading to the formation of abasic sites 14 rather than to DNA strand scission. The mechanism (eqs 7, 8, and 10 in Scheme 2) is also consistent with the simultaneous formation of SSB, abasic sites, and Cr(III)-DNA complexes during the DNA reactions with Cr(VI) in the presence of GSH or ascorbate (2).

Inhibition of DNA Cleavage by H Donors and Conditions for the Formation of Cr(III)-DNA Complexes. The observed inhibition of Cr(V)-ehba-induced DNA cleavage by organic compounds (Figures 6-8) can be explained in terms of repair reactions (44) of DNA radicals 3 and 6 (eq 9 in Scheme 2). The use of acetate buffers, as well as of Cr(V) stock solutions in DMSO [which are much more stable than those in water (40)], was the reason for the failures in previous works (8, 15) to detect DNA cleavage induced by Na[CrVO(ehba)2] at pH >6. The ability of organic molecules to react with DNA radicals is likely to be connected with the electronic stabilization of the product radicals (an example for DMSO is shown in eq 9 of Scheme 2). The proposed H-abstraction mechanism (44) is supported by a decrease in the inhibition levels when CD3COONa is used instead of CH3COONa (Figure 6). As DMSO is a much more efficient inhibitor of DNA cleavage at pH 7.0 than acetate (compare Figures 6 and 7), the repair reaction (eq 9 in Scheme 2) of DNA radicals with DMSO is probably too fast to affect the overall rate of DNA cleavage. This explains the absence of changes in inhibition levels when DMSO is replaced with DMSO-d6 (Figure 7). The absence of significant Cr(V)-induced DNA cleavage in Tris or HEPES buffers (Figure 1) is explained by H-donor properties of these buffers (Figure 8).

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DNA cleavage at higher pH values (7.0 vs 4.0) was more sensitive to the presence of either of the H donors (Figures 6 and 7) and O2 (Figure 5). This is probably because DNA radicals 3 and 6 are more readily formed and/or are more stable at higher pH values. The H donors and O2 then compete for radicals 3 and 6 (eqs 7 and 9 in Scheme 2). On the other hand, the intermediates 4 and 11, which are unable to react either with H donors or with O2, are more likely to be formed at lower pH values. The reactions of H donors with C4′-radicals of doublestranded DNA lead to regeneration of natural DNA with high stereoselectivity, thus eliminating DNA damage (44). The repair reactions, similar to eq 9 in Scheme 2, are expected to inhibit the DNA cleavage caused by Cr(V) and -(IV) species in intracellular media, which are rich in potential H donors. However, the formation of kinetically inert Cr(III)-DNA complexes (13) is possible even in the presence of inhibitors (eq 9 in Scheme 2). Binding of Cr(III) has been shown to alter the biological functions of DNA (42, 46). Thus, the formation of Cr(III)-DNA complexes during the reactions of Cr(V) and -(IV) with DNA is likely to be more damaging in vivo than the DNA strand breaks caused by Cr(V) and -(IV), as the latter are easily eliminated by the presence of H donors (44). Effects of Ligand Concentrations on DNA Reactivities with Cr(V) versus Cr(IV). The work described here presents the first direct evidence that the wellcharacterized (21-25) Cr(IV) 2-hydroxycarboxylato complexes cleave plasmid DNA in vitro. The results [Figures 10, 11, and S1-S3 (Supporting Information)] confirm the assumption (22) that the Cr(IV) 2-hydroxycarboxylates are more likely than the corresponding Cr(V) complexes to cleave DNA in the presence of excess ligand. Sugden and Wetterhahn (11) suggested that Cr(IV)-ehba complexes formed during the disproportionation of Na[CrVO(ehba)2] (in acetate buffers, at pH 5.0-7.0, with no excess ligand added) are the ultimate reacting species in the oxidation of thymidine nucleotides. This suggestion arose from the observation that the oxidation was inhibited by Mn(II), which catalyzes the disproportionation of Cr(IV)ehba (47), but does not affect that of Cr(V)-ehba (48). However, an alternative explanation of this inhibition is possible, namely, the Mn(II) reduction of the reactive intermediates, such as nucleotide radicals (R•) and peroxo radicals (ROO•), formed in the reactions of nucleotides (RH) with Cr(V) in the presence of O2. The results of this work suggest that the plasmid DNA cleavage can be preferentially initiated by either Cr(IV) or Cr(V) complexes, dependent on the reaction conditions. DNA cleavage by Na[CrVO(ehba)2] at pH 4.0 is completely inhibited by excess ligand at concentrations as low as 1 mM (Figure 9). This is consistent with the strong shift of the equilibrium (eq 2 in Scheme 1) toward bis-chelated species Ia (6, 7, 22), while the Cr(IV)-ehba complexes exist mainly in the monochelated form IIb at pH 4.0 and ehba concentrations of 1 min at an initial Cr(IV) concentration of 0.50 mM and 37 °C, as determined by UV/vis spectroscopy.

Levina et al.

pH >6.5 (Figures 10 and S2), though these Cr(IV) species were relatively stable up to pH 7.5 (22).8 The possible reasons for the lack of DNA cleavage by Cr(IV) in neutral media are (i) Cr(IV) complexes IIb may require protonation to be converted into an active form and (ii) at high pH values, the excess ligand may act as an alkyl H donor (eq 9 in Scheme 2). Thus, Cr(V) monochelates Ib are the most likely DNA cleaving species in the absence of added ligand, while the role of Cr(IV) monochelates IIb increases with an increase in the ligand concentration.

Acknowledgment. Financial support of this work from the Australian Research Council, the National Health and Medical Research Council of Australia, the Cancer Research Fund of the University of Sydney, an Australian National University Collaborative Research Grant, and a CSIRO Collaborative Research Grant is gratefully acknowledged. We thank Mrs. Penelope Lilley (Australian National University) for preparation of plasmid DNA. Supporting Information Available: Figures showing the reactivities of plasmid DNA with Cr(V) and -(IV) 2-hydroxycarboxylato complexes: comparison of Cr(V)- and -(IV)-ehba, Cr(V)- and -(IV)-qa and control systems; pH dependencies for the reactions of Cr(V)- and -(IV)-qa at a qa concentration of 50 mM; and qa concentration dependencies at pH 5.0. This material is available free of charge via the Internet at http:// pubs.acs.org.

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