Disproportionation of a model chromium(V) complex causes extensive chromium(III)-DNA binding in vitro.

The first direct evidence for the role of Cr(V) complexes in the formation of potentially mutagenic Cr(III)-DNA adducts has been obtained. A model complex for the stabilized Cr(V) species formed in Cr(VI)-treated cells, [Cr(V)O(ehba)(2)]-[ehba = 2-ethyl-2-hydroxybutanoato(2-)], rapidly disproportionates in HEPES buffers at pH 7.4 [3 Cr(V) --> 2 Cr(VI) + Cr(III)], and the formed Cr(III) species undergo efficient ionic binding to DNA, followed by slower covalent binding. The extent of Cr(III)-DNA binding significantly exceeds that caused by [Cr(III)(OH(2))(6)](3+) or by the Cr(III) products of Cr(VI) reductions under similar conditions. The Cr(III)-DNA binding can be dramatically reduced by the ability of the reaction medium (e.g., phosphate buffer) to form complexes with Cr(III) during and after the disproportionation reaction. A mechanism of Cr(III)-DNA binding caused by Cr(V) disproportionation has been proposed on the basis of stoichiometric and kinetic studies.

Chromium(VI) reduction by catechol(amine)s results in DNA cleavage in vitro: relevance to chromium genotoxicity.

Catechols are found extensively in nature both as essential biomolecules and as the byproducts of normal oxidative damage of amino acids and proteins. They are also present in cigarette smoke and other atmospheric pollutants. Here, the interactions of reactive species generated in Cr(VI)/catechol(amine) mixtures with plasmid DNA have been investigated to model a potential route to Cr(VI)-induced genotoxicity. Reduction of Cr(VI) by 3,4-dihydroxyphenylalanine (DOPA) (1), dopamine (2), or adrenaline (3) produces species that cause extensive DNA damage, but the products of similar reactions with catechol (4) or 4-tert-butylcatechol (5) do not damage DNA. The Cr(VI)/catechol(amine) reactions have been studied at low added H(2)O(2) concentrations, which lead to enhanced DNA cleavage with 1 and induce DNA cleavage with 4. The Cr(V) and organic intermediates generated by the reactions of Cr(VI) with 1 or 4 in the presence of H(2)O(2) were characterized by EPR spectroscopy. The detected signals were assigned to Cr(V)-catechol, Cr(V)-peroxo, and mixed Cr(V)-catechol-peroxo complexes. Oxygen consumption during the reactions of Cr(VI) with 1, 2, 4, and 5 was studied, and H(2)O(2) production was quantified. Reactions of Cr(VI) with 1 and 2, but not 4 and 5, consume considerable amounts of dissolved O(2), and give extensive H(2)O(2) production. Extents of oxygen consumption and H(2)O(2) production during the reaction of Cr(VI) with enzymatically generated 1 and N-acetyl-DOPA (from the reaction of Tyr and N-acetyl-Tyr with tyrosinase, respectively) were correlated with the DNA cleaving abilities of the products of these reactions. The reaction of Cr(VI) with enzymatically generated 1 produced significant amounts of H(2)O(2) and caused significant DNA damage, but the N-acetyl-DOPA did not. The extent of in vitro DNA damage is reduced considerably by treatment of the Cr(VI)/catechol(amine) mixtures with catalase, which shows that the DNA damage is H(2)O(2)-dependent and that the major reactive intermediates are likely to be Cr(V)-peroxo and mixed Cr(V)-catechol-peroxo complexes, rather than Cr(V)-catechol intermediates.

EPR spectroscopic studies of the reduction of chromium(VI) by methanol in the presence of peptides. Formation of long-lived chromium(V) peptide complexes.

The synthesis and characterization of the first Cr(V) complexes with non-sulfur-containing peptides, which may mimic the chemistry of the intermediates in the formation of Cr-induced peptide-DNA cross-links in vivo, are reported. The reduction of Cr(VI) with methanol in the presence of a number of non-sulfur-containing peptides produced relatively stable Cr(V)-peptide complexes, which were characterized by EPR spectroscopy and electrospray mass spectrometry. The reaction of Cr(VI) with methanol alone (in the absence of peptide ligands) resulted in the formation of two Cr(V)-methanol intermediates, with giso values of 1.9765 and 1.9687. The methanol reduction of Cr(VI) in the presence of the glycine peptides, triglycine, tetraglycine, and pentaglycine resulted in the formation of both Cr(V)-methanol and Cr(V)-peptide intermediates, while only the Cr(V)-peptide complexes were detected in the reactions with the alanine peptides trialanine, tetraalanine, and pentaalanine. Similar EPR signals were observed for all of the Cr(V)-peptide complexes with giso values between approximately 1.986 and approximately 1.979, and AN values of (2.1-2.6) x 10(-4) cm-1.

An investigation of the chromium oxidation state of a monoanionic chromium tris(catecholate) complex by X-ray absorption and EPR spectroscopies.

The well-known monoanionic Cr tris(3,5-di-tert-butylcatecholato) complex, [Cr(DTBC)3]-, has been studied by X-ray absorption spectroscopy. The multiple-scattering fit to the XAFS gave good correlation (R = 19.8%) and good values for all of the bond lengths, angles, and Debye-Waller factors. The principal bond lengths and angles around the metal center (Cr-O, 1.96 A; O-C, 1.28 A; O-Cr-O, 81.8 degrees; Cr-O-C, 113.3 degrees) were most consistent with the XRD structure for [Cr(X4C6O2)3]- (X = Cl, Br), compared to those in other oxidation states, [Cr(DTBC)3], [Cr(Cl4C6O2)3], and [Cr(O2C6H4)3]3-. The XANES spectrum shows the main K edge at 6003.3 eV and a preedge peak at 5992.9 eV, which is approximately 8% of the intensity of the main K edge. The XANES data were compared to those for Cr-ehba complexes (ehbaH2 = 2-ethyl-2-hydroxybutanoic acid) of known oxidation states (III, IV, and V) and show, in conjunction with EPR spectroscopy and a reevaluation of XRD structures and theoretical calulations, that the complex is best described as a Cr(V) center with delocalization from the catechol ligands. The [Cr(catecholato)3]n+ (n = 1, 0) complexes have similar EPR spectroscopic and structural properties, respectively, to the 1- complex and are also best described as Cr(V) complexes. Such intermediates are important in the redox reactions of catechol(amine)s, and oxidized amino acids (e.g., DOPA), with carcinogenic Cr(VI) and may have relevance in Cr-induced cancers.

Permeability, cytotoxicity, and genotoxicity of Cr(III) complexes and some Cr(V) analogues in V79 Chinese hamster lung cells.

The permeabilities and genotoxicities of the Cr(III) complexes [Cr(en)(3)](3+), mer-[Cr(glygly)(2)](-), cis-[Cr(phen)(2)(OH(2))(2)](3+), and trans-[Cr(salen)(OH(2))(2)](+) and the Cr(V) analogues of cis-[Cr(phen)(2)(OH(2))(2)](3+) and trans-[Cr(salen)(OH(2))(2)](+) [en being 1,2-ethanediamine, glygly being glycylglycine, phen being 1,10-phenanthroline, and salen being N,N'-ethylenebis(salicylideneiminato)] have been studied in V79 Chinese hamster lung cells. Following exposure of approximately 10(6) cells to 0.4 mM Cr(III) for 4 h, the Cr uptake by single cells was less than 10(-)(14) g/cell (as determined by GFAAS analysis and as confirmed by PIXE analysis where the Cr concentration was below the limit of detection). Importantly, the Cr(V) analogue of cis-[Cr(phen)(2)(OH(2))(2)] was significantly more permeable than the Cr(III) complex. The cytotoxicity of the Cr(III) complexes increased in the following order: mer-[Cr(glygly)(2)](-) < [Cr(en)(3)](3+) approximately cis-[Cr(phen)(2)(OH(2))(2)](3+) < trans-[Cr(salen)(OH(2))(2)](+). No genotoxic effects were observed following exposure to mer-[Cr(glygly)(2)](-) or [Cr(en)(3)](3+) at concentrations up to 6 mM. The Cr(III) imine complexes trans-[Cr(salen)(OH(2))(2)](+) and cis-[Cr(phen)(2)(OH(2))(2)](3+), which could be oxidized to Cr(V) complexes, induced MN in vitro at rates of 13.6 and 3.3 MN/1000 BN cells/micromol of Cr, respectively. The comparative permeabilities and genotoxicities of trans-[Cr(salen)(OH(2))(2)](+) and [CrO(salen)](+) were similar due to the instability of the Cr(V) complex at physiological pH values (7.4). There was a substantial increase in the permeability of [Cr(O)(2)(phen)(2)](+), compared to that of the Cr(III) analogue, which was accompanied by a highly genotoxic response. Consequently, any Cr(III) complex that is absorbed by cells and can be oxidized to Cr(V) must be considered as a potential carcinogen. This has potential implications for the increased use of Cr(III) complexes as dietary supplements and highlights the need to consider the genotoxicities of a variety of Cr(III) complexes when determining the carcinogenic potential of Cr(III) particularly when "high" deliberately administered doses are concerned.

Disproportionation and nuclease activity of bis[2-ethyl-2-hydroxybutanoato(2-)]oxochromate(V) in neutral aqueous solutions.

Complex 1, [Cr(V)O(ehba)2]- (ehba = 2-ethyl-2-hydroxybutanoate(2-)) is the most studied model compound of relevance to the biological activity of Cr(V) with regard to Cr-induced cancers. The first detailed kinetic study of disproportionation of 1 under neutral pH conditions (pH 6.0-8.0, [NaClO4] = 1.0 M, 37 degrees C) is reported. Kinetic data were collected by stopped-flow and conventional UV-vis spectroscopies and processed by the global analysis method. The disproportionation, which follows the stoichiometry 3Cr(V) --> 2Cr(VI) + Cr(III) (1), leads to release of 5 mol of H+/3 mol of Cr(V). Reaction 1 is accelerated by phosphate, but is not affected by acetate, HEPES, or Tris buffers. Initial rates of Cr(V) decay are directly proportional to [Cr(V)]0 (0.020-1.0 mM); they increase with an increase in the pH values and decrease in the presence of a large excess of ehba ligand. The first direct evidence for the formation of Cr(IV) intermediates in reaction 1 has been obtained; however, their UV-vis spectral properties were different from those of the well-characterized Cr(IV)-ehba complexes. The Cr(III) products of reaction I in phosphate buffers differ from those in the other buffers. A mechanism is proposed for reaction 1 on the basis of kinetic modeling. Influences of the reaction time and conditions on the extent of plasmid DNA cleavage induced by 1 have been studied under conditions corresponding to those of the kinetic studies. A comparison of the kinetic and DNA cleavage results has shown that direct interaction of 1 with the phosphate backbone of DNA is the most likely first step in the mechanism of DNA cleavage in neutral media. Small additions of Mn(II) ((0.01-0.1)[Cr(V)]0) did not affect the rate and stoichiometry of reaction 1, but suppressed the formation of Cr(IV) intermediates (presumably due to the catalysis of Cr(IV) disproportionation). However, much higher concentrations of Mn(II) ((0.1-1.0)[Cr(V)]0) were required to inhibit DNA cleavage induced by 1. Thus, contrary to previous reports (Sugden, K. D.; Wetterhahn, K. E. J. Am. Chem. Soc. 1996, 118, 10811-10818), inhibition by Mn(II) does not indicate a key role of Cr(IV) in Cr(V)-induced DNA cleavage.

EPR studies of chromium(V) intermediates generated via reduction of chromium(VI) by DOPA and related catecholamines: potential role for oxidized amino acids in chromium-induced cancers.

The reductions of K2Cr2O7 by catecholamines, DOPA, DOPA-beta,beta-d2, N-acetyl-DOPA, alpha-methyl-DOPA, dopamine, adrenaline, noradrenaline, catechol, 1,2-dihydroxybenzoic acid (DHBA), and 4-tert-butylcatechol (TBC), produce a number of Cr(V) electron paramagnetic resonance (EPR) signals. These species are of interest in relation to the potential role of oxidized proteins and amino acids in Cr-induced cancers. With excess organic ligand, all of the substrates yield Cr species with signals at g(iso) approximately 1.972 (Aiso(53Cr) > 23.9 x 10(-4) cm(-1)). These are similar to signals reported previously but have been reassigned as octahedral Cr(V) species with mixed catechol-derived ligands, [CrV(semiquinone)2(catecholate)]+. Experiments with excess K2Cr2O7 show complex behavior with the catecholamines and TBC. Several weak Cr(V) signals are detected after mixing, and the spectra evolve over time to yield relatively stable substrate-dependent signals at g(iso) approximately 1.980. These signals have been attributed to [Cr(O)L2](L = diolato) species, in which the Cr is coordinated to two cyclized catecholamine ligands and an oxo ligand. Isotopic labeling studies with DOPA (ring or side chain deuteration or enrichment with 15N), and simulation of the signals, show that the superhyperfine couplings originate from the side chain protons, confirming that the catecholamine ligands are cyclized. At pH 3.5, a major short-lived EPR signal is observed for many of the substrates at g(iso) approximately 1.969, but the species responsible for this signal was not identified. Several other minor Cr signals are detected, which are attributed (by comparison with isoelectronic V(IV) species) to Cr(V) complexes coordinated by a single catecholamine ligand (and auxiliary ligands e.g. H2O), or to [Cr(O)L2]- (L = diolato) species with a sixth ligand (e.g. H2O). Addition of catalase or deoxygenation of the solutions did not affect the main EPR signals. When the substrates were in excess (pH > 4.5), primary and secondary (cyclized) semiquinones were also detected. Semiquinone stabilization by Zn(II) complexation yielded stronger EPR signals (g(iso) approximately 2.004).

Characterization and X-ray absorption spectroscopic studies of bis[quinato(2-)]oxochromate(V).

A new Cr(V) complex, K[CrVO(qaH3)2].H2O (Ia; qaH3 = quinato = (1R,3R,4R,5R)-1,3,4,5-tetrahydroxycyclohexanecarboxylato(2-)), synthesized by the reaction of K2Cr2O7 with excess qaH5 in MeOH (Codd, R.; Lay, P. A. J. Am. Chem. Soc. 1999, 121, 7864-7876), has been characterized by microanalyses, electrospray mass spectra, and UV-visible, CD, IR, EPR, and X-ray absorption spectroscopies. This complex is of interest because of its ability to act as both a structural and a biomimetic model for a range of Cr(V) species believed to be generated in vivo during the intracellular reduction of carcinogenic Cr(VI). The Na+ analogue of Ia (Ib) has also been isolated and characterized by microanalyses and IR and X-ray absorption spectroscopies. The reaction of Cr(VI) with MeOH in the presence of qaH5 that leads to I proceeds via a Cr(IV) intermediate (observed by UV-visible spectroscopy), and a mechanism for the formation of I has been proposed. DMF or DMSO solutions of I are stable for several days at 25 degrees C, while I in aqueous solution (pH = 4) disproportionates to Cr(VI) and Cr(III) in minutes. The likely structures in the solid state for Ia (14 K) and Ib (approximately 293 K) have been determined using both single-scattering (Ia,b) and multiple-scattering (Ia) analyses of XAFS data. These analyses have shown the following: (i) In agreement with the results from the other spectroscopic techniques, the quinato ligands are bound to Cr(V) by 2-hydroxycarboxylato moieties, with Cr-O bond lengths of 1.55, 1.82, and 1.94 A for the oxo, alcoholato, and carboxylato O atoms, respectively. (ii) The position of an oxo O atom is somewhat disordered. This is consistent with molecular mechanics modeling of the likely structures. The XAFS, EPR, and IR spectroscopic evidence points to the existence of hydrogen bonds between the oxo ligand and the 3,4,5-OH groups of the quinato ligands in the solid state of I.

Permeability, cytotoxicity, and genotoxicity of chromium (V) and chromium (VI) complexes in V79 Chinese hamster lung cells.

The genotoxicity of Cr(V) complexes in mammalian cells (V79 Chinese hamster lung cells) has been studied for the first time using the in vitro micronucleus assay. Two complexes were investigated, [CrO(ehba)2]-, which undergoes ligand-exchange and disproportionation reactions in the cell growth medium, and [CrO(mampa)]-, which is chemically inert in the medium for the duration of the exposure period. Results of in vitro micronucleus assays show that both complexes are genotoxic and exhibit similar potencies to that of [Cr2O7]2-. The permeabilities of the Cr(V) complexes were also investigated for the first time using particle-induced X-ray emission (PIXE) analysis of individual cells. The Cr uptake increased in the order: [Cr(phen)2-(H2O)2]3+ < [CrO(ehba)2]- < [CrO(mampa)]- < [Cr2O7]2-. Clonal assays showed that Cr(VI) exhibits an expectedly higher cytotoxicity than the Cr(V) complexes. While the genotoxicities of the Cr(V) and Cr(VI) complexes increase according to their permeabilities, the genotoxicities of the Cr(V) complexes are equal to, if not greater than, that of Cr(VI) in terms of the amount of Cr entering the cell. This supports other evidence that Cr(V), produced as a metabolic intermediate from the intracellular reduction of Cr(VI), may be important in Cr-induced cancers.

Microprobe X-ray absorption spectroscopic determination of the oxidation state of intracellular chromium following exposure of V79 Chinese hamster lung cells to genotoxic chromium complexes.

The oxidation state of intracellular chromium has been determined directly in mammalian lung cells exposed to mutagenic and carcinogenic chromium compounds. Microprobe X-ray absorption spectroscopy (XAS) experiments on single V79 Chinese hamster lung cells showed that Cr(VI) and Cr(V) complexes were reduced completely (>90%) to Cr(III) within 4 h of exposure of the cells. This result provides direct evidence for the hypothesis that these genotoxic oxidants react rapidly with intracellular reductants.

In vitro DNA damage and mutations induced by a macrocyclic tetraamide chromium(V) complex: implications for the role of Cr(V) peptide complexes in chromium-induced cancers.

Electron paramagnetic resonance and electronic absorption spectroscopies have shown that unlike the bidentate Cr(V) complex [Cr(ehba)2O]- (ehba = 2-hydroxy-2-ethylbutanoato(2-)), I, the macrocyclic tetradentate complex, [Cr (mampa-dcb)(O)]- (mampa-dcb = 5,6-(4,5-dichlorobenzo)-3,8,11,13-tetraoxo-2,2,9,9-tetrameth yl-12,12-diethyl-1, 4,7,10-tetraazacyclotridecane), II, is substitutionally inert. Low levels of DNA strand cleavage were observed after treatment with II under physiological conditions (50 mM sodium phosphate, pH 7.4, 37 degrees C) at concentrations as high as 2 mM for periods as long as 2 days. II also induces a lower number of revertants in mutation assays with Salmonella typhimurium TA100 than I when identical Cr concentrations are applied. The slopes of the linear portion of the dose-response curves are parallel, however, indicating that the mutagenicity of II is comparable to I. II is stable toward ligand exchange, reduction and disproportionation in the mutagenicity test medium and also in the presence of bacteria and the common cell reductant, glutathione. This indicates that ligand exchange with DNA and/or reduction to Cr(IV) are not responsible for the mutagenicity of II (unlike I). It is believed that II reversibly but weakly intercalates with DNA placing the Cr(V) center in close proximity for hydrogen atom abstraction or oxo-transfer reactions to ensure. This tetraamide complex is a good structural and biomimetic model for non-sulfur-containing Cr(V) peptide species that may form in vivo from reactions of Cr(VI) with peptides. Hence, it is likely to be relevant to understanding one possible mechanism by which Cr(VI) causes cancer.