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J. Am. Chem. Soc. 2000, 122, 6208-6216
Reactions of Chromium(VI/V/IV) with Bis(O-ethyl-L-cysteinato-N,S)zinc(II): A Model for the Action of Carcinogenic Chromium on Zinc-Finger Proteins1 Aviva Levina, Angela M. Bailey, Guillaume Champion,† and Peter A. Lay* Contribution from the School of Chemistry, UniVersity of Sydney, Sydney, 2006 New South Wales, Australia ReceiVed December 16, 1999
Abstract: The reactions of Cr(VI/V/IV) with a model thiolato complex [Zn(SR)2] (RSH ) O-ethyl-L-cysteine), resembling a tetrahedral (2S,2N) Zn(II) binding site in zinc-finger proteins, have been studied in comparison with those of the free ligand. The stability of [Zn(SR)2] in aqueous solutions with pH 6-11 (25 °C, Arsaturated) has been established by CD spectroscopy. Stoichiometries and products of the reactions of [Zn(SR)2] or RSH with [CrVIO4]2- or [CrVO(ehba)2]- (ehba ) 2-ethyl-2-hydroxybutanoato(2-)) at pH 6.5-8.5 (25 °C, Ar) were studied by UV-vis and CD spectroscopies and by electrospray mass spectrometry. Disulfide (RSSR) is the only detectable oxidation product of both [Zn(SR)2] and RSH. In the case of RSH, relatively stable Cr(III) complexes, [CrIII(SR)3]0 and [CrIII(SR)2]+, are also formed and subsequently hydrolyzed to [CrIII(Cys)2]-. This is the first observation of a Cr(III)-facilitated hydrolysis of a cysteine ester. Ascorbate promotes the oxidation of [Zn(SR)2] by [CrVIO4]2-; this reaction leads to formation of RSSR and of the Cr(III)thiolato complexes. Kinetics of the reactions of [Zn(SR)2] or RSH with [CrVIO4]2-, [CrVO(ehba)2]-, or [CrIVO(qa)(qaH)]- (qa ) quinato(2-) ) (1R,3R,4R,5R)-1,3,4,5-tetrahydroxycyclohexanecarboxylato(2-)) at pH 7.40 (25 °C, Ar) were studied by conventional and stopped-flow UV-vis spectrophotometry and by global kinetic analysis. Complexes of Cr(V) and Cr(IV) rapidly react with both the reductants, and reactions of Cr(V) pass through the Cr(IV) intermediates. By contrast with the reaction with RSH, the reaction of [CrVO(ehba)2]- with [Zn(SR)2] is not accompanied by a significant O2 consumption (measured by an oxygen electrode), suggesting the ability of [Zn(SR)2] to inhibit free radical reactions, similar to that of zinc metallothioneins. The mechanism of [Zn(SR)2] oxidation by Cr(VI/V/IV), including intramolecular formation of a disulfide bond, has been proposed. Implications of the results to the Cr(VI)-induced carcinogenesis and to the biological activity of Cr(III) are discussed.
Introduction Chromium(VI) is an established carcinogen and one of the major chemical occupational hazards.2 Mutagenicity of Cr(VI) in cell assays is well-known, but mechanisms of this action are still a matter of debate.3 At the current state of knowledge, the following chemical properties of Cr are thought to be responsible for its mutagenicity and carcinogenicity: (i) tetrahedral [CrVIO4]2ions (resembling [SO4]2- and [HPO4]2-) easily permeate the cells through anion channels,4 while insoluble Cr(VI) particles enter the cells by phagocytosis;5 (ii) inside the cells or while †
On leave from Ecole Normale Supe´rieure de Lyon, 69007 Lyon, France. (1) Abbreviations: Cys ) L-cysteinato(2-); DMF ) dimethylformamide; DMSO ) dimethyl sulfoxide; EDTA ) ethylenediamine-N,N,N′,N′tetraacetic acid; ehbaH2 ) 2-ethyl-2-hydroxybutanoic acid; ESMS ) electrospray mass spectrometry; HEPES ) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; qaH2 ) D-(-)-quinic acid ) (1R,3R,4R,5R)-1,3,4,5tetrahydroxycyclohexanecarboxylic acid; RSH ) O-ethyl-L-cysteine; Tris ) tris(hydroxymethyl)aminomethane. (2) IARC. Monographs on the EValuation of the Carcinogenic Risk of Chemicals to Humans. Vol. 49. Chromium, Nickel and Welding; International Agency on the Research of Cancer: Lyon, France, 1990. (3) De Flora, S.; Camoirano, A.; Bagnasco, M.; Zanacchi, P. In Handbook of Metal-Ligand Interactions in Biological Fluids; Berthon, G., Ed.; Marcel Dekker: New York, 1995; Vol. 2, pp 1020-1036. (4) Connett, P. H.; Wetterhahn, K. E. Struct. Bonding (Berlin) 1983, 54, 93-124. (5) Singh, J.; Carlisle, D. L.; Pritchard, D. E.; Patierno, S. R. Oncol. Rep. 1998, 5, 1307-1318 and references therein.
passing through cell membranes, Cr(VI) is reduced by a variety of compounds, such as ascorbate, glutathione, NADH, and tocopherols,6 causing the formation of the highly reactive Cr(V/IV) and free radical intermediates, which are capable of inducing oxidative damage to DNA and proteins;4-6 (iii) Cr(V/IV) can exist in the cells as relatively long-lived species, stabilized by biological ligands such as 2-hydroxycarboxylates and 1,2-diolates;7 (iv) kinetically inert Cr(III) complexes8 of DNA and proteins are formed as the final products of Cr(VI) intracellular reductions, leading to the lesions that may disrupt functioning of the biomolecules.9 Numerous studies have shown the abilities of the Cr(VI) + reductant systems and of the model Cr(V/IV) complexes to induce various forms of potentially carcinogenic DNA damage, including strand breaks, abasic sites, and DNA-protein or DNA-DNA cross-links.10,11 (6) Cies´lak-Golonka, M. Polyhedron 1996, 15, 3667-3689 and references therein. (7) Codd, R.; Lay, P. A. J. Am. Chem. Soc. 1999, 121, 7864-7876 and references therein. (8) Larkworthy, L. F.; Nolan, K. B.; O’Brien, P. In ComprehensiVe Coordination Chemistry; Wilkinson, G., Gillard, R. D., McCleverty, J. A., Eds.; Pergamon Press: Oxford, U.K., 1987; Vol. 3, pp 699-969. (9) (a) Zhitkovich, A.; Voitkun, V.; Kluz, T.; Costa, M. EnViron. Health Perspect. 1998, 106 (Suppl. 4), 969-974. (b) Bridgewater, L. C.; Manning, F. C. R.; Patierno, S. Mol. Carcinog. 1998, 23, 201-206 and references therein.
10.1021/ja9944047 CCC: $19.00 © 2000 American Chemical Society Published on Web 06/16/2000
Reactions of Cr(VI/V/IV) with a Thiolato Complex Another possible mechanism of Cr genotoxicity, namely, the Cr(VI)-induced damage of nuclear transcription factors, has received much less attention. Disruption of transcription factor functions, induced by transition-metal ions, can block the normal DNA replication,12 or prevent the repair of damaged DNA sites.13 Zinc-finger proteins form the largest family of transcription factors; some of them, such as p53, are thought to be critically important in carcinogenesis.14 Biological activity of zinc-finger proteins is dependent on the formation of Zn(II)-S bonds with the protein cysteine residues, and oxidative damage of these bonds leads to the loss of protein-DNA binding ability.15 There are several conflicting reports on suppression16 or activation17 of the DNA binding abilities of different transcription factors in the presence of Cr(VI/V/IV). Our preliminary studies have shown a decrease in DNA binding ability of a zinc-finger protein, GATA-1, treated by a model Cr(V) complex.18 The chemistry of Cr(VI/V/IV) interactions with zinc-finger proteins remains unknown due to the absence of model studies and to the general lack of knowledge on the reactivities of Zn(II)-S bonds.19 In this work, we studied the reactions of Cr(VI/V/IV) with a simple model complex, bis(O-ethyl-L-cysteinato-N,S)zinc(II) ([Zn(SR)2]), which mimics a typical tetrahedral (2Cys, 2His) binding site of Zn(II) in zinc-finger proteins, in comparison with the corresponding reactions of the free ligand (RSH). The relatively stable complexes, [CrVO(ehba)2]- and [CrIVO(qa)(qaH)]-, used in this work are models of stabilized Cr(V/IV) species which are likely to form intracellularly.7 The results demonstrate the ability of Cr(VI/V/IV) to cause Zn(II) release with the formation of S-S bonds, and to a lesser extent, of Cr(III)-S bonds upon the reaction with [Zn(SR)2]. The ability of [Zn(SR)2] to inhibit the formation of reactive oxygen species, resembling that of zinc metallothioneins,20 has also been established. In addition, hydrolysis of RSH has been observed to be facilitated by binding to Cr(III); this reaction may be relevant to Cr(III)-peptide interactions in vivo. (10) (a) Kortenkamp, A.; Casadevall, M.; Da Cruz Fresco, P.; Shayer, R. O. J. NATO ASI Ser., Ser. 2 1997, 26, 15-34. (b) Stearns, D. M.; Wetterhahn, K. E. NATO ASI Ser., Ser. 2 1997, 26, 55-72 and references therein. (11) Levina, A.; Barr-David, G.; Codd, R.; Lay, P. A.; Dixon, N. E.; Hammershøi, A.; Hendry, P. Chem. Res. Toxicol. 1999, 12, 371-381 and references therein. (12) Louie, A. Y.; Meade, T. J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6663-6668 and references therein. (13) Hartwig, A. Toxicol. Lett. 1998, 102-103, 235-239. (14) (a) Klug, A. J. Mol. Biol. 1999, 293, 215-218. (b) Green, A.; Parker, M.; Conte, D.; Sarkar, B. J. Trace Elem. Exp. Med. 1998, 11, 103-118. (c) Me´plan, C.; Mann, K.; Hainaut, P. J. Biol. Chem. 1999, 274, 3166331670 and references therein. (15) (a) Casadevall, M.; Sarkar, B. J. Inorg. Biochem. 1998, 71, 147152. (b) Fojta, M.; Kubicarova, T.; Vojtesek, B.; Palecek, E. J. Biol. Chem. 1999, 274, 25749-25755. (c) Park, J. S.; Wang, M.; Park, S. J.; Lee, S. H. J. Biol. Chem. 1999, 274, 29075-29080. (16) Shumilla, J. A.; Wetterhahn, K. E.; Barchowsky, A. Arch. Biochem. Biophys. 1998, 349, 356-362. (17) (a) Ye, J. P.; Zhang, X.; Young, H. A.; Mao, Y.; Shi, X. Carcinogenesis 1995, 16, 2401-2405. (b) Shi, X. L.; Ding, M.; Ye, J. P.; Wang, S. W.; Leonard, S. S.; Zang, L. Y.; Castranova, V.; Vallyathan, V.; Chiu, A.; Dalal, N.; Liu, K. J. J. Inorg. Biochem. 1999, 75, 37-44. (c) Kaltreider, R. C.; Pesce, C. A.; Ihnat, M. A.; Lariviere, J. P.; Hamilton, J. W. Mol. Carcinog. 1999, 25, 219-229. (18) (a) O’Connell, A. M. B.Sc. (Hons.) Thesis, University of Sydney, 1997. (b) Bailey, A. M.; Crossley, M.; Lay, P. A. Unpublished results. (19) (a) Kuehn, C. G.; Isied, S. S. Prog. Inorg. Chem. 1980, 27, 153221. (b) Brand, U.; Rombach, M.; Vahrenkamp, H. J. Chem. Soc., Chem. Commun. 1998, 2717-2718 and references therein. (20) (a) Elgohary, W. G.; Sidhu, S.; Krezoski, S. O.; Petering, D. H.; Byrnes, R. W. Chem.-Biol. Interact. 1998, 115, 85-107. (b) Cai, L.; Tsiapalis, G.; Cherian, M. G. Chem.-Biol. Interact. 1998, 115, 141-151. (21) Leonard, A.; Lauwerys, R. R. Mutat. Res. 1980, 75, 49-62 and references therein.
J. Am. Chem. Soc., Vol. 122, No. 26, 2000 6209 Experimental Section Caution. Cr(VI) and As(III) compounds are human carcinogens,2,21 and Cr(V/IV) complexes are mutagenic and potentially carcinogenic.22 Contact with skin and inhalation must be aVoided. Reagents. The following commercial reagents of analytical or higher purity grade were used without purification: L-cysteine, O-ethyl-Lcysteine hydrochloride, DMF, DMSO, 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent), 2-ethyl-2-hydroxybutanoic acid, As2O3, NaOH, and Na2CrO4‚4H2O (all Aldrich); CH3COOH, HClO4, HCl (35% aqueous solution), NH3 (32% aqueous solution), Na2EDTA, and KBr (all Merck); Zn(SO4)2‚7H2O (BDH Chemicals); L-ascorbic acid (BDH Biochemicals); D-(-)-quinic acid (ICN Biomedicals); Tris and Chelex 100 (Bio-Rad); HEPES (Research Organics); sym-diphenylcarbazide (Fluka). Water was purified by the Milli-Q technique. Zinc(II) complexes, [Zn(SR)2] and Na2[Zn(Cys)2]‚6H2O, were synthesized by literature methods,23 and their purities were confirmed by FTIR spectroscopy and by determination of [Zn] and [thiol]. Chromium complexes, Na[CrVO(ehba)2]‚H2O and Na[CrIII(Cys)2]‚2H2O, were synthesized by literature methods,24,25 and their purities were confirmed by FTIR spectroscopy (Cr(V) and Cr(III)), UV-vis and EPR spectroscopies (Cr(V)), or determination of [Cr] and [thiol] (Cr(III)). General Methods. Stock solutions of the buffers (0.50 M HEPES + NaOH, pH 7.40, or 1.0 M CH3COOH + NH3, pH 7.4) were treated by Chelex 100 chelating resin (the pH values were measured after the treatment using an Activon 210 ionometer with an AEP 321 combined glass/calomel electrode), and stored at 4 °C. Concentrations of the catalytic metals (Fe(III) and Cu(II)) in the purified buffers, determined by Buettner’s ascorbate method,26 were 7.41,56 Thus, Zn(II) can either catalyze or inhibit the reactions of Cr(VI) with thiols, dependent on the nature of substrate and on the pH value and the buffer conditions. Hydrolysis of Cr(III)-Bound Cysteine Ester. It is known that the metal ions with the high affinities to the thiol group (Ni(II), Cd(II), Zn(II), Pb(II), Hg(II)) promote the alkaline hydrolysis of cysteine esters.42 In the current work, it was found that Cr(III) is an effective promotor of RSH hydrolysis in a slightly basic medium (pH 8.5, 25 °C). By contrast, significant hydrolysis of Zn(II)-bound RSH was observed only at pH g 11 (25 °C). A possible mechanism of the Cr(III)-assisted hydrolysis of RSH is presented in Scheme 4. Two main Cr(III)-RSH complexes, [CrIII(SR)3]0 and [CrIII(SR)2]+ (detected by ESMS, Figure 4a and Table 1), exist in solutions in the presence of excess RSH (eq 10 in Scheme 4). The latter species is easily converted to the thermodynamically more stable25,39 [CrIII(Cys)2]- complex (eq 11 in Scheme 4); this reaction is driven by the high affinity of Cr(III) for carboxylato donors.8 Complexes of Cr(III) with cysteine-rich peptides are known to act as cofactors in the interaction of insulin molecules with cell (54) Yu, X.; Hathout, Y.; Fenselau, C.; Sowder, R. C.; Henderson, L. E.; Rice, W. G.; Mendeleyev, J.; Kun, E. Chem. Res. Toxicol. 1995, 8, 586-590. (55) (a) Perez-Benito, J. F.; Lamrhari, D.; Arias, C. Can. J. Chem. 1994, 72, 1637-1644. (b) Perez-Benito, J. F.; Saiz, N.; Amat, E. J. Mol. Catal., A 1998, 135, 1-10. (56) Dı´az-Cruz, M. S.; Mendieta, J.; Monjonell, A.; Tauler, R.; Esteban, M. J. Inorg. Biochem. 1998, 70, 91-98.
6216 J. Am. Chem. Soc., Vol. 122, No. 26, 2000 Scheme 4. Proposed Mechanism for the Hydrolysis of Cr(III)-Bound RSH
LeVina et al. peptide complexes by providing the required geometries of the insulin-Cr(III)-peptide-receptor adducts. Conclusion Studies of the model systems have shown the susceptibility of Zn(II)-S bonds to the oxidation by Cr(VI/V/IV) species at physiological pH values. Similar reactions in the organisms exposed to Cr(VI) may cause the oxidative damage of Zn(II)dependent transcription factors (zinc-finger proteins), leading to the disruption of normal DNA replication, and contribute to the Cr(VI)-induced carcinogenesis. Acknowledgment. The financial support of this work by an Australian Research Council (ARC) grant (to P.A.L.) and ARC RIEFP grants for the EPR, CD, and stopped-flow spectrometers is gratefully acknowledged. We thank Dr. Merlin Crossley (Department of Biochemistry, University of Sydney) for helpful discussions. Supporting Information Available: (i) Figures showing the correlation between [CrIIISR] values and the intensities of CD signals, typical experimental and simulated signals in ESMS, and the results of global kinetic analyses and (ii) tables showing the detailed results of stoichiometry, product, and kinetic studies (PDF). This material is available free of charge via the Internet at http://pubs.acs.org. JA9944047
membrane receptors.57 Reactions similar to eqs 10 and 11 (Scheme 4) may contribute to the biological activity of Cr(III)-
(57) (a) Davis, C. M.; Sumrall, K. H.; Vincent, J. B. Biochemistry 1996, 35, 12963-12969. (b) Davis, C. M.; Vincent, J. B. Arch. Biochem. Biophys. 1997, 339, 335-343.
