Importance of Chromium−DNA Adducts in Mutagenicity and Toxicity of

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Chem. Res. Toxicol. 2005, 18, 3

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Review Importance of Chromium-DNA Adducts in Mutagenicity and Toxicity of Chromium(VI) Anatoly Zhitkovich Department of Pathology and Laboratory Medicine, Brown University, Box G-E507, Providence, Rhode Island 02912 Received August 12, 2004

1. Introduction 2. Aqueous Chemistry of Chromium(III) and Chromium(VI) 2.1. Hexavalent Chromium 2.2. Trivalent Chromium 3. Role of Cr-DNA Adducts in DNA Damage by Chromium(VI) 3.1. Metabolism of Cr(VI) by Biological Reducers 3.2. Major Types of Cr-DNA Adducts 3.3. Effects of Cr-DNA Adducts on Replication in Vitro 3.4. Mutagenic Role of Cr-DNA Adducts 3.5. Nucleotide Specificity of Cr Mutagenesis and DNA Binding 3.6. Cr-DNA Adducts and Toxicity of Chromium(VI) 4. Mechanisms of Cr-DNA Damage and Qualitative Risk Assessment Modeling for Chromium(VI) 5. Summary

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1. Introduction Several million industrial workers worldwide are potentially exposed to Cr and Cr-containing compounds (1, 2). Environmental contamination with Cr has also become a significant concern due to continuous industrial emissions and the presence of heavily contaminated sites in the vicinity of residential areas. Cr exists in many chemical and physical forms that vary in their environmental behavior and biological activity. Cr(0), Cr(III), and Cr(VI) are three main oxidative states of chromium that are most commonly found in the workplace and general environment. Cr(0) is typically encountered in its metallic form in alloys with other metals, such as Ni, Fe, and Co. Stainless steel is the largest volume product containing metallic Cr. Although Cr(0) is stable to oxidation by atmospheric oxygen under ambient conditions, high temperature processes such as welding or exposure to corrosive chemicals lead to the formation of higher oxidation states, Cr(III) and Cr(VI). Cr-based prosthesis can also undergo slow oxidation with the resulting release * To whom correspondence should be addressed. Tel: 401-863-2912. Fax: 401-863-9008. E-mail: [email protected].

of higher oxidative forms of Cr (3, 4). Only Cr(VI)containing compounds are generally found to cause toxic effects in exposed people. Workers in approximately 70 professional groups experience occupational exposure to different Cr(VI) compounds. The highest exposure to the toxic Cr(VI) form is usually detected in the chrome plating industry, among chromate production workers and stainless steel welders (1, 2). Metallic Cr is chemically and biologically inert, but exposure to Cr(0)containing dust may cause nonspecific irritation in the respiratory tract. Exposure to various forms of hexavalent Cr compounds has been consistently found to be associated with an elevated incidence of respiratory cancers and other adverse health consequences (5-9). The most common form of cancer found among Cr(VI)-exposed workers is squamous cell carcinoma of the lung (10). The genotoxicity of Cr(VI) has been confirmed in animal experiments and in numerous cellular assays that measured mutagenic and clastogenic activities (2, 11, 12). Exposure to Cr(VI)-containing acid mists in electroplating industry leads to ulceration and perforation of nasal septum and impairs lung functions (12). Other health consequences of inhalation exposure to Cr(VI) include pulmonary fibrosis, chronic bronchitis, emphysema, and bronchial asthma (8, 9). Ingestion of large doses of Cr(VI) can result in the irritation of mucous membranes and, in severe cases, intestinal bleeding and renal tubular necrosis. In contrast to Cr(VI), epidemiological studies have found no association between the exposure to Cr(III) compounds and the risk of cancer (1, 2, 7). The lack of carcinogenic activity of inorganic Cr(III) complexes is further supported by negative results in animal experiments and cellular genotoxicity tests (12). Low toxicity of Cr(III) compounds is generally attributed to their poor uptake and low retention by the cells (11). At physiological conditions, Cr(VI) by itself is unreactive toward DNA and requires reductive activation to cause biological damage. The reduction process is associated with the production of unstable Cr(V), Cr(IV), organic radicals, and finally yields Cr(III) (14-17). The main attention in the field of Cr(VI) toxicology for many years has been focused on a potential role of reactive oxygen species, which was supported by studies on the induction of oxidative stress or DNA oxidation in the presence of supraphysiological concentrations of Cr(VI) (for review, see ref 18). However, intracellular reduction

10.1021/tx049774+ CCC: $30.25 © 2005 American Chemical Society Published on Web 12/15/2004

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Figure 1. Main aqueous forms of chromium(VI).

of Cr(VI) has also been known to lead to the formation of stable Cr-DNA complexes (19). The biological significance of these complexes was initially difficult to assess because of their unknown composition. In vitro studies that used phosphate buffers to avoid radical scavenging effects of their organic counterparts have failed to detect Cr-DNA binding during the reduction of Cr(VI) by biological reducers (for example, studies with thiols; 20, 21), and this was considered as indicative of the predominant role of oxidative mechanisms in DNA damage by Cr(VI). However, it has recently been determined that phosphate buffer is an inappropriate medium to analyze Cr-DNA interactions due to the formation of unreactive Cr(III)-phosphate complexes (22). The production of various Cr-DNA adducts during Cr(VI) reduction is now well-established, and this review will discuss the biological significance of these lesions, mechanisms of their generation, and potential implications for Cr(VI) risk assessment modeling.

2. Aqueous Chemistry of Chromium(III) and Chromium(VI) 2.1. Hexavalent Chromium Cr(VI) is strongly oxidizing, which severely limits a number of its compounds, as the majority of potential ligands will be oxidized with the accompanied reduction of the metal to lower oxidation states. As a result, Cr(VI) is present almost exclusively in complexes with halide and oxygen ligands. Chromium trioxide (CrO3), chromyl chloride (CrO2Cl2), and various chromates are the most commonly encountered Cr(VI) compounds. Depending on the pH and metal concentrations of the solution, Cr(VI) can exist as chromate (CrO42-), hydrochromate (HCrO4-), or dichromate (Cr2O72-) ion (Figure 1). Chromate is the main anionic form at neutral pH (about 75%), while lowering the pH below 6 leads to the predominant presence of HCrO4- (23, 24). In acidic solutions with Cr(VI) concentrations above 10 mM, HCrO4- forms a dimer, Cr2O72-. At physiological pH and typical Cr(VI) exposure levels, the formation of dichromate ion is expected to be negligible. Chromate has a tetrahedral arrangement of oxygen groups, which makes it structurally similar to sulfate and phosphate and permits its easy entry into the cells via the general anion channel (25, 26). Once inside the cell, Cr(VI) is rapidly reduced to Cr(III) that forms stable complexes with proteins and DNA and this leads to a very high intracellular accumulation of the metal (25). Cr(VI) is thermodynamically stable in a wide range of pH values when dissolved in pure water. However, it is easily reduced in highly acidic solutions containing any organic molecules with oxidizable groups, including DNA and proteins. At neutral pH, Cr(VI) is much more difficult to reduce and only a few biological molecules have been

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found to exhibit significant rates of Cr(VI) reduction (27). The strong oxidizing ability of Cr(VI) at low pH is caused by its very high positive E0 value at these conditions, which permits electron transfer reactions from a broad range of organic donors. Even for agents that are capable of reducing Cr(VI) at pH 7 (for example, glutathione), the rate of Cr(VI) reduction gets progressively higher at low pH (28), which reflects a more favorable formation of esters between the reducer and the HCrO4-. In light of the ability of many organic molecules to reduce Cr(VI) in highly acidic solutions, the reported values for Cr(VI) reducing capacity by different biological preparations (29, 30) should be considered with caution. These studies relied on the direct measurements of residual Cr(VI) in the calorimetric reaction with diphenylcarbazide in the presence of 8% sulfuric acid, conditions that likely overestimated the reducing capabilities of the biological systems. More accurate determinations of Cr(VI) reductive activities require the removal of organic molecules by charcoal or other means prior to the addition of the diphenylcarbazide reagent. A strong pH dependence of Cr(VI) reduction has important implications for the understanding of biological effects of Cr(VI) exposure via different routes. Because of favorable conditions for the reduction of Cr(VI) in the acidic environment of the stomach, oral intake of low and moderate doses of Cr(VI) by experimental animals has been generally found to cause more limited or no toxicity (30-32) and relatively low tissue levels of total chromium (33).

2.2. Trivalent Chromium Thermodynamically stable Cr(III) forms six-coordinate complexes with H2O and many other molecules that contain O, N, or S atoms. Cr(III) acts as a hard Lewis acid and strongly prefers coordination to negatively charged oxygen groups. All Cr(III) complexes have octahedral arrangements of ligands. There are many hundreds if not thousands of various mononuclear Cr(III) complexes that have been produced for various applications. The complexity of Cr(III)-ligand interactions is evident even in simple aqueous solutions of Cr(III). Freshly prepared unbuffered solutions of inorganic Cr(III) salts contain hexaaquachromium(III) [Cr(H2O)63+] as the major species (Figure 2A). These solutions undergo rapid hydrolysis associated with the conversion of Cr(H2O)63+ into Cr(OH)(H2O)52+ and Cr(OH)2(H2O)5+ (34). The pKa of Cr(H2O)63+ has not been precisely determined and is believed to be about 4. The formation of the Cr(III) hydroxo species initiates polymerization reactions that yield dimer, trimer, and higher polymeric forms (Figure 2B). Monomeric and low oligomeric forms of Cr(III) are usually soluble whereas polymeric products form precipitates. The hydrolytic polymerization of Cr(III) is strongly influenced by “aging” time, pH, concentration, and matrix composition of the solutions (35, 36). Surface waters typically contain a mixture of soluble monomeric and multinuclear Cr(III) species. Adjustment of Cr(H2O)63+-containing solutions to pH 7 results in almost instantaneous formation of insoluble Cr(III) hydroxides and polynuclear species. Because of the solubility and oligomerization problems, the reactions of inorganic Cr(III) with DNA or proteins have been frequently performed at pH 6 or lower (21, 37). Unlike Cr(H2O)63+, stable complexes of Cr(III) with

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Figure 2. Forms of chromium(III) in aqueous solutions. (A) Mononuclear species and (B) polynuclear species.

multidentate organic ligands, such as amino acids or picolinate, are soluble at neutral pH and do not undergo oligomerization. A rapid coordination with the reducer is responsible for maintaining the solubility of Cr(III) produced after reduction of Cr(VI) at physiological pH (38). Interestingly, preparation of chromium(III) chloride in HEPES buffers with neutral pH apparently avoids the massive formation of poorly soluble Cr(III) hydroxo species, as Cr(III) avidly reacts with DNA under these conditions (39). There is evidence of significant Cr(III)HEPES interactions in neutral solutions (18), and the formation of these complexes probably accounts for the solubility of Cr(III) in this buffer. Other organic buffers containing negatively charged sulfonate groups, such as MOPS and MES, are also capable of some binding to Cr(III) (22), which is expected to inhibit the formation of insoluble hydroxides. Cr3+ does not interact with neutral or positively charged Tris molecules, and the solubility and reactivity of Cr(III) in low molarity Tris buffers usually reflect the inability of these solutions to maintain neutral pH in the presence of strongly acidic Cr(H2O)63+ (40). Acidification of DNA solutions can also result in the generation of Cr(III) adduct-unrelated damage primarily in the form of abasic sites created by the loss of the guanine base. The lack of Cr(III) toxicity has generally been attributed to the inability of Cr3+-containing complexes to enter cells. While much lower cellular concentrations of Cr after exposure to Cr(III) complexes relative to chromate are not in question, the description of Cr(III) compounds as universally incapable of entering cells does not appear to be correct. Even excluding experiments with cultured cells when Cr(III) precipitates could be formed and then taken up via phagocytosis, there is strong evidence from in vivo studies demonstrating the cellular uptake of Cr(III) (4, 41-43). The inability of Cr(III) complexes to enter blood cells in many in vitro experiments can also be associated with the use of citratestabilized blood because citrate is a strong Cr(III) chelator. Cr(III) does not accumulate in blood cells in vivo and appears to exist in equilibrium between extracellular and

Figure 3. Chemical structures of the major biological reducers of chromium(VI).

intracellular compartments. The ability of certain Cr(III) complexes to enter the cell does not necessarily mean that toxic effects should ensue as coordination with multidentate ligands makes Cr(III) unreactive (9).

3. Role of Cr-DNA Adducts in DNA Damage by Chromium(VI) 3.1. Metabolism of Cr(VI) by Biological Reducers Reductive metabolism of Cr(VI) inside the cell or in vitro always yields Cr(III) as the final oxidative form. Low molecular weight thiols (glutathione and cysteine) and ascorbate are believed to be primarily responsible for the intracellular reduction of Cr(VI) (44-47). Chemical structures of the main biological reducers of Cr(VI) are shown in Figure 3. Studies on the reduction of Cr(VI) by extracts from rat lung, liver, or kidney have found that ascorbate accounted for at least 80% of Cr(VI) metabolism in these target tissues (45, 46). Ascorbate is also the fastest reducer of Cr(VI) in the in vitro reactions, and its rate of reduction at 1 mM concentration exceeds that of cysteine and glutathione approximately 13 and 61 times, respectively (48). Depending on the nature of the reducing agent and its concentration, this process can

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generate various amounts of unstable Cr(V) and Cr(IV) intermediates (14-16). Reductive reactions with the twoelectron reducer ascorbate yield Cr(IV) as the first reaction intermediate when ascorbate is present at a 2:1 or greater molar excess over Cr(VI) (15, 17, 49). The presence of Cr(V) was only detectable in reactions of Cr(VI) at nonphysiological conditions employing equimolar or lower concentrations of ascorbate. The formation of Cr(V) under conditions of limited ascorbate concentrations probably occurs through the reactions of comproportionation or disproportionation of Cr(IV) (CrVI + CrIV f 2CrV or 2CrIV f CrV + CrIII, respectively). The initial step in reduction of Cr(VI) by physiological concentrations of cysteine proceeds primarily through oneelectron transfer (47), while the glutathione-driven reaction is largely a two-electron process (50). The final product of Cr(VI) reduction, Cr(III), forms stable adducts with macromolecules and other cellular constituents. In the in vitro reactions, Cr(III) is usually coordinated with two molecules of the unoxidized reducer, such as ascorbate or cysteine (38, 51).

3.2. Major Types of Cr-DNA Adducts The ability to form stable complexes with many ligands and the presence of six coordination sites gives Cr(III) the opportunity to generate various DNA cross-links with other molecules (ternary adducts). Ternary DNA crosslinks formed by Cr(III)-mediated bridging of glutathione, cysteine, histidine, and ascorbate represent the major form of Cr-DNA adducts in Cr(VI)-exposed mammalian cells (52, 53). Cysteine-Cr(III)-DNA and glutathioneCr(III)-DNA cross-links were the most abundant DNA modifications detected following exposure of CHO cells to Cr(VI) and comprised 24 and 17% of all Cr-DNA adducts, respectively (52). Importantly, CHO cells were grown in ascorbate-containing RMEM medium in these studies. All ternary DNA adducts are formed through the attachment of Cr(III) to DNA phosphates (Figure 4) (37, 53). Glutathione- and amino acid-Cr-DNA cross-links were estimated to account for about 50% of total DNAbound Cr without corrections for recovery (52). Ascorbate-Cr-DNA cross-links were recovered with about 40% efficiency (53), and if this value is roughly applicable to other ternary complexes, then practically all cellular Cr-DNA adducts are ternary cross-links. Reductive metabolism of Cr(VI) in vitro usually generates a large number of binary Cr(III)-DNA adducts (22, 37, 53), but the presence of these DNA modifications in cells has not yet been established. The formation of binary Cr-DNA complexes in cells is expected to be strongly inhibited due to the abundance of intracellular ligands capable of rapid coordination to Cr(III) prior to its binding to DNA. This suggestion is supported by the findings on a shift toward the formation of ternary cross-links in reactions containing high concentrations of Cr(III)-coordinating ligands, such as histidine or ascorbate (37, 53). The presence of ascorbate-Cr(III)-DNA adducts in human A549 cells was only detected after restoration of physiological concentrations of ascorbic acid in these cells prior to Cr(VI) exposure (53). Under standard tissue culture conditions, A549 and many other human and rodent cells either lack detectable ascorbate or contain it only at micromolar levels (physiological levels are in millimolar range) (53, 54) due to low concentrations of this vitamin in fetal bovine serum and its absence in the most

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commonly used types of growth media (DMEM, RPMI 1640, F10, F12). The level of ascorbate in cultured cells depends on its concentration in serum and the length of time after the last change of medium. One day old cultures typically contain about 50 µM ascorbate (54) while its levels are practically undetectable after 2 days of growth (53). The presence of variable concentrations of ascorbate could be an important factor in the biological variability of Cr(VI) experiments with cultured cells. DNA-protein cross-links have also been detected in various Cr(VI)-treated cells (55, 56) and tissues in vivo (57, 58) and can be readily generated during Cr(VI) reduction in vitro (19). Although these lesions represent only a very small fraction of initially formed DNA adducts in cultured cells (about 0.1% according to our calculations), the availability of a robust methodology for their measurements (58) and poor repair abilities of peripheral blood lymphocytes (59, 60) have led to the use of DNAprotein cross-links as a biomarker of genetic damage in Cr-exposed human populations (61-64). About 50% of DNA-protein cross-links isolated from Cr(VI)-treated CHO cells were disrupted by incubation in the presence of EDTA demonstrating that this fraction of the lesions is formed by Cr(III)-dependent bridging of DNA and proteins (65). The EDTA labile portion of DNA-protein cross-links purified from Cr(VI)-treated human lymphoblastoid MOLT4 cells was much smaller, which led to the suggestion that the majority of cross-links were probably formed via oxidative mechanisms (66). Because the spectra of Cr(VI)- and oxidant-cross-linked proteins are completely different (65-68), it is possible that a low reversibility of DNA-protein cross-links by EDTA is caused by a generally poor ability of this chelator to dissociate DNA-bound Cr(III) even when it was present in the form of sterically more accessible small adducts (22). Characterization of the chemistry of DNA-protein cross-links is expected to provide the basis for the development of analytical methodologies for the measurements of these lesions with better Cr specificity (9). In vitro reduction of Cr(VI) by ascorbate (39, 69) or cysteine (22), but not by glutathione (70), also produces a small number of Cr(III)-mediated interstrand DNA cross-links. The formation of interstrand cross-links was highly dependent on the ratio of reducer to Cr(VI), and the most extensive DNA cross-linking was always observed under conditions of limited reducer concentrations. For cysteine-based reactions, the yield of cross-links averaged 1% but was much smaller or even undetectable at low Cr(VI) concentrations (22, 71). DNA cross-linking in Cr(VI)-ascorbate reactions followed a similar trend, with minimal or no cross-linking occurring in the presence of 5-fold or higher molar excess of the reducer (39, 69). While there is more than one pair of sites on the opposite DNA strands that can be potentially linked by Cr(III) (18), the formation of interstrand cross-links by Cr(III) atom is difficult to explain from the steric point of view. To link two DNA strands, a highly polar and bulky octahedral Cr(III) complex should intercalate between DNA bases, which is normally observed only for nonpolar planar or linear molecules. On the basis of the steric considerations and the fact that the yield of interstrand cross-links had the exponential dose dependence (22), it is possible that DNA cross-linking is caused by oligomeric Cr(III) species, not monomeric Cr(III). In this scenario, the opposite DNA strands are linked by multinuclear Cr(III) complexes that wrap around the

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DNA duplex and bind each strand via different Cr(III) atoms. Blocking of DNA cross-linking in Cr(VI) reactions containing severalfold molar excess of ascorbate (39), which still generates about 75% binary Cr(III)-DNA adducts (53), is also in agreement with the possibility that Cr(III) oligomers are the cross-linking species.

3.3. Effects of Cr-DNA Adducts on Replication in Vitro Increased misincorporation of nucleotides by prokaryotic polymerases during in vitro replication of DNA templates with a low number of binary Cr(III)-DNA adducts (72) and the induction of arrest sites during replication of more heavily modified Cr(III)-DNA templates (73) provided the first evidence pointing to the biological significance of Cr-DNA adducts. Replicationarresting potential of a subset of Cr(III)-DNA adducts was also detected in DNA templates modified by Cr(VI) in the presence of limited concentrations of ascorbate (69) and was subsequently observed using purified mammalian DNA polymerases R and β (74). Polymerase arrest sites were preferentially observed at the positions one nucleotide upstream of guanine residues, but templates with high numbers of adducts showed more uniform distribution of replication-blocking damage (73). A strong correlation between the presence of interstrand DNA cross-links and the polymerase inhibition led to the conclusion that these lesions were likely responsible for the blockage of DNA replication in vitro (39, 69). Recent studies have eliminated the possibility that polymerase arrests in DNA templates treated with Cr(VI)/ascorbate were caused by oxidative damage (39).

3.4. Mutagenic Role of Cr-DNA Adducts The ability of various Cr-DNA adducts to induce mutations in human cells was examined using a shuttlevector approach. In this methodology, a specific type of Cr-DNA adduct can be selectively formed and then tested for its ability to cause mutations and inhibit replication in cells. Ternary Cr-DNA adducts formed by cross-linking of glutathione or amino acids have been found to be mutagenic following replication of a shuttlevector plasmid in human SV40-immortalized fibroblasts (75). Among peptide/amino acid cross-links, the glutathione-Cr(III)-DNA cross-link was the most mutagenic and there was a strong trend for the bulkiness of the cross-linked ligand to be associated with a greater mutagenic potency of Cr-DNA complexes. Binary Cr(III)-DNA adducts were only weakly mutagenic, and their mutagenic potential was much lower as compared to any ternary Cr-DNA cross-link (48, 75, 76). The ascorbate-Cr(III)-DNA cross-link, the most recently characterized ternary DNA adduct (53), was also found to be highly mutagenic in human fibroblasts (48). On the basis of the mutagenicity normalized to that of binary Cr(III)-DNA adduct, the ascorbate-Cr-DNA cross-link appears to be the most potent mutagenic form of CrDNA modifications. DNA-protein cross-links have not yet been tested for their mutagenic activity; however, the size dependence of mutagenesis by amino acid-Cr(III)DNA adducts (75) points to a potentially strong mutagenic potential of protein-containing DNA lesions. Alternatively, the extreme bulkiness of DNA-protein cross-links would cause only replication arrests and no

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mutagenesis. The mutagenic potential of Cr(III)-formed interstrand DNA cross-links has not yet been examined either, and these lesions are probably largely genotoxic (replication blocking) but not mutagenic. This suggestion is supported by findings on a much higher yield of mutations after replication of Cr(III)-modified plasmids in XP-A human fibroblasts (77), which are deficient in the removal of Cr-DNA adducts but have normal repair of interstrand DNA cross-links. As Cr(VI) metabolism can potentially generate many forms of DNA damage, including oxidative lesions, studies have also been performed to determine the overall importance of Cr adduct-dependent and oxidative pathways in the mutagenicity and genotoxicity of Cr(VI) activated by different biological reducers. Dissociation of DNA-bound Cr(III), as well as blocking of Cr-DNA adduction by Cr(III) chelators or by shielding of DNA phosphates by Mg2+ ions, have all been found to eliminate mutagenic and replication blocking damage in plasmids reacted with Cr(VI) in the presence of cysteine (76) or ascorbate (48). These results demonstrated the critical importance of Cr-DNA adducts in the DNA-damaging activity of Cr(VI) in the reactions with two reducers that were estimated to account for 80-95% of Cr(VI) reductive metabolism in the target tissues (45, 46, 48). The relative contribution of Cr-DNA adducts to DNA damage produced in the reactions of Cr(VI) with glutathione has not yet been determined, but it is likely to be very significant considering a high mutagenic potential of glutathioneCr-DNA cross-links (75). Consistent with the important mutagenic role of Cr-DNA adducts, mutational spectra of Cr(VI) and hydrogen peroxide in human TK6 cells were found to be very different (78), indicating that nonoxidative mechanisms were largely responsible for the induction of mutagenesis by Cr(VI) in vivo.

3.5. Nucleotide Specificity of Cr Mutagenesis and DNA Binding Single base substitutions were the predominant type of mutations induced by glutathione- and amino acidCr(III)-DNA adducts (75). On average, point mutations constituted approximately 80% of all mutations induced by these adducts during replication of shuttle-vector plasmids in human fibroblasts. Ascorbate-Cr-DNA cross-link showed a significantly different distribution of types of mutations, which was characterized by almost equal frequency of point mutations and larger genetic changes (48). A higher yield of deletions in ascorbate cross-link-containing plasmids was probably caused by a stronger replication-blocking potential of this adduct in human cells (48). Single base changes induced by all Cr-DNA adducts targeted the G/C pair leading to G/CfT/A transversions and G/CfA/T transitions (48, 75, 76). The G/CfT/A transversions were generally more common events, and their frequency was particularly high for the largest adduct, glutathione-Cr-DNA crosslink. Mutational changes caused by Cr-DNA adducts were similar to those observed in Cr(VI)-treated cells in which the mutational spectrum was dominated by G/CfT/A and G/CfA/T substitutions (78). Studies on Cr(III) binding to mononucleotides and oligonucleotides of base specific composition have shown that metal coordination occurs primarily at the phosphate group (37, 53). Nucleotide level mapping of binary and ternary Cr-DNA adducts along the supF gene detected

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Figure 4. Proposed structure of cysteine-Cr(III)-DNA crosslink. This adduct is the most abundant Cr-DNA modification in Cr(VI)-treated cells (52). Tridentate coordination of cysteine to Cr(III) has been determined by analysis of crystal structures of bis(L-cysteinato)chromium(III) complexes (85, 86).

Figure 6. Formation mechanisms and biological effects of Cr-DNA adducts. Asc, ascorbate; Cys, cysteine; GSH, glutathione; and L, ligand (ascorbate, cysteine, glutathione, or histidine).

Figure 5. Formation of microchelates (intramolecular crosslinks) by DNA-bound chromium(III). (A) Direct coordination of Cr(III) to 5′-phosphate and N-7 of dG. This mode of DNA binding is likely to be limited to small binary Cr(III)-DNA adducts (76). (B) Direct coordination of Cr(III) to 5′-phosphate and hydrogen bonding to N-7 of dG. This binding mode can occur for both binary and ternary Cr(III)-DNA adducts.

no base specificity in Cr binding; however, these adducts caused G/C-targeted mutations at this gene (75). It has been proposed that Cr(III) binding to 5′-phosphate of dG allows additional coordination to N-7 of this base (Figure 5), and the formation of this microchelate (intramolecular cross-link) causes greater duplex distortions and G selective mutagenesis (76).

3.6. Cr-DNA Adducts and Toxicity of Chromium(VI) In addition to the induction of mutations, ternary DNA adducts have also been found to possess a significant genotoxic potential as detected by highly reduced yields

of replicated molecules following propagation of adducted plasmids in human fibroblasts (48, 76). Small binary Cr(III)-DNA adducts were only weakly genotoxic in the same assay. Decreased template activity of Cr(III)modified DNA appears to largely reflect the presence of interstrand DNA cross-links as suggested by in vitro replication experiments using Taq polymerase (39) and a similar yield of replicated plasmids in normal and nucleotide excision repair deficient XP-A cells (77). Nucleotide excision repair has been found to be the main mechanism for removal of Cr-DNA adducts in human cells, and the inactivation of this repair pathway through the loss of the XPA protein in human fibroblasts or lung epithelial H460 cells led to much higher apoptosis and clonogenic death after Cr(VI) exposure (77). Because XPA deficient cells apparently have normal repair of DNAprotein (60) and interstrand DNA cross-links (79, 80), these results provide genetic evidence for the importance of Cr-DNA adducts in cytotoxic effects of Cr(VI). High genotoxic potential in cellular replication assays (48, 76) and its dependence on the XPA status (77) all support the idea that ternary Cr-DNA adducts are a major form of toxic DNA modifications in Cr(VI)-treated cells. Interestingly, ternary Cr-DNA adducts do not significantly impede in vitro replication of adducted templates with purified polymerases (39, 70), suggesting that cytotoxic and replication-blocking activities of these DNA modifications in vivo reflect complex cellular responses rather than a simple block for replicating enzymes.

4. Mechanisms of Cr-DNA Damage and Qualitative Risk Assessment Modeling for Chromium(VI) All Cr-DNA adducts contain trivalent Cr, which is the final thermodynamically stable oxidative state of this metal in cells. There are two potential mechanisms for the formation of Cr(III)-DNA adducts: one based on the direct DNA binding by Cr(III) and another involving initial binding to DNA by intermediate Cr(V,IV) forms followed by the completion of reduction to Cr(III). The

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formation of insoluble Cr(III) hydroxides at neutral pH (21, 40) and generally slow rates of ligand exchange reactions by Cr(III) complexes (81) have been two main arguments that Cr(III) is not a DNA reactive form in Cr(VI) reduction reactions. As discussed above, Cr(III) remains soluble in many neutral solutions that contain ligands other than water, such as amino acids, biological buffers with ionized sulfonate groups, and, more generally, almost any multidentate ligands. Freshly prepared inorganic Cr(III) complexes have been consistently shown to be very reactive in forming Cr(III)-DNA adducts (37, 48, 73-75). The production of Cr-DNA adducts has also been found to continue even after the completion of Cr(VI) reduction by cysteine (76) or ascorbate (37, 48, 53), demonstrating the DNA binding ability of Cr(III). Thus, these findings indicate that direct reaction of Cr(III) with DNA is at least one of the major mechanisms of the generation of binary Cr-DNA adducts. For ternary CrDNA adducts, the role of Cr(III) is critical as these adducts are formed by attack of DNA by binary Cr(III)ligand complexes (37, 53). Because of the need for the initial generation of Cr(III)-cysteine complexes, the formation of cysteine-Cr(III)-DNA cross-links lags the production of binary Cr-DNA adducts during reduction of Cr(VI) (76). While it is possible that a fraction of binary Cr(III)DNA adducts is also formed through the mechanism involving the initial DNA coordination with higher oxidative forms of Cr, this process is not dependent on the presence of Cr(V). Human cells in vivo contain millimolar concentrations of ascorbate (82), which means that this reducer will be present in a large molar excess over gradually entering Cr(VI) even in the cases of heavy exposure. Reduction of Cr(VI) by ascorbate under these conditions produces Cr(IV) as the main intermediate form and no detectable Cr(V) (14, 15, 43). Nevertheless, Cr-DNA adducts are rapidly and extensively formed in these reactions (37, 39, 48, 53), indicating that Cr(V) is not needed for the generation of Cr-DNA modifications. The absence of Cr(V)-DNA and Cr(V)-mononucleotide ESR signals in the reactions with high concentrations of Cr(V) complexes with NADPH (83) or 2-ethyl-2-hydroxybutanoic acid (84) demonstrated the overall poor potential of Cr(V) for DNA binding. Mechanistic considerations of the formation of Cr-DNA lesions have potentially important implications for Cr(VI) risk assessment modeling regarding DNA damage deposition at different exposure levels. If Cr(V) were the critical DNA binding form, then highly nonlinear or even threshold models of DNA damage formation can be invoked, as this Cr form is produced only at concentrations of Cr(VI) approaching those of ascorbate. As discussed above, experimental evidence demonstrated that the reaction of Cr(III) complexes with DNA is the principal mechanism for the formation of the most abundant and toxicologically important ternary Cr-DNA cross-links. This reaction mechanism predicts that the formation of Cr-DNA adducts is largely independent of the reducing capacity of the cell and should generate essentially a linear yield of DNA damage relative to the intracellular dose of Cr(VI) at occupationally or environmentally relevant exposure levels. This prediction is supported by the measurements of Cr-DNA adducts following exposure of human lung epithelial H460 cells to low concentrations of Cr(VI) (77). The linear model for Cr-DNA binding argues against a threshold mechanism

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in genetic damage by intracellular Cr(VI), but it does not exclude the possibility of a nonlinear cancer risk due to the importance of various biological processes in malignant transformation of Cr-damaged cells. For example, a carcinogenic process involving selection for mismatch repair deficient cells that are resistant to Cr(VI) toxicity (87) should produce nonlinear dose dependence. Comprehensive risk modeling also needs to take into account the role of detoxification processes associated with extracellular reduction of Cr(VI).

5. Summary Intracellular reduction of Cr(VI) results in the extensive formation of Cr-DNA adducts, among which Cr(III)mediated DNA cross-links of glutathione, cysteine, histidine, and ascorbate represent a major class of DNA modifications. Cysteine-Cr(III)-DNA and glutathioneCr(III)-DNA cross-links are the two most abundant lesions that collectively account for about 80% of all ternary Cr-DNA adducts in cultured cells. In vitro reduction of Cr(VI) also generates binary Cr(III)-DNA adducts, but their significant production in cells is uncertain. All ternary and a major fraction of binary Cr(III)-DNA adducts are formed by attack of DNA by Cr(III) complexes. The formation mechanisms for the relatively rare DNA-protein and interstrand DNA crosslinks have not yet been determined. All ternary Cr-DNA adducts were mutagenic during replication in human cells. The mutagenic and genotoxic potentials of Cr-DNA adducts were related to the bulkiness of the attached ligand, with large glutathione- and ascorbate-Cr(III)DNA cross-links generating the highest yields of mutants. Cr-DNA adducts were responsible for all mutagenic and replication-blocking damage generated during reduction of Cr(VI) with cysteine or ascorbate, which are two main reducers of Cr(VI) in vivo. Nucleotide excision repair has been identified as the principal mechanism for removal of Cr-DNA adducts in human cells. The strong genotoxic potential of all main adducts and hypersensitivity of nucleotide excision repair deficient human cells to apoptosis and clonogenic death indicate that Cr-DNA adducts are also a significant cause of Cr(VI) toxicity. Thus, it appears that Cr-DNA adducts are key genetic lesions contributing to the induction of the main biological effects of Cr(VI), such as mutagenesis, replication inhibition, and cell death (Figure 6). The formation of genotoxic ternary Cr-DNA adducts is predicted to be independent of the reducing capacity of the cell and should exhibit linear dose dependence with respect to intracellular Cr(VI).

Acknowledgment. I acknowledge support from the National Institute of Environmental Health Sciences by Grants ES008786 and ES012915.

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