Chem. Res. Toxicol. 1997, 10, 533-535
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Communications 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 Carolyn T. Dillon,† Peter A. Lay,*,† Marian Cholewa,*,‡,§ George J. F. Legge,*,‡ Antonio M. Bonin,| Terrence J. Collins,⊥ Kimberley L. Kostka,⊥ and Grace Shea-McCarthy# School of Chemistry, University of Sydney, NSW 2006, Australia, Micro Analytical Research Centre (MARC), School of Physics, University of Melbourne, Parkville, Victoria 3052, Australia, Institute of Nuclear Physics, Cracow, Poland, Worksafe Australia, P.O. Box 58, Sydney, NSW 2001, Australia, Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, and CARS, University of Chicago at National Synchrotron Light Source, Brookhaven National Laboratory, Upton, New York 11973 Received January 27, 1997X
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.
Introduction Cr(VI) complexes are human carcinogens, and Cr(V) has been established as a potential carcinogen (1-10). Previously, we have shown that a proton-induced X-ray emission (PIXE)1 microprobe could be used to measure the uptake and intracellular distribution of Cr compounds by individual cultured mammalian V79 Chinese hamster lung cells (11).2 These and other experiments involving analysis of bulk cells (12-16) showed a 10200-fold concentration of Cr when the cells were exposed to Cr(V) and Cr(VI) complexes. This concentration of Cr in the cells was presumed to be due to rapid reduction to the less permeable Cr(III), which would create a concentration gradient and drive the accumulation of high Cr concentrations in the cell. The rapid reduction of high oxidation states to Cr(III) was also postulated in the uptake-reduction model of Wetterhahn (17). * Authors to whom correspondence should be addressed at the University of Sydney (P.A.L.) or the University of Melbourne (M.C. or G.J.F.L.). † University of Sydney. ‡ University of Melbourne. § Institute of Nuclear Physics. | Worksafe Australia. The views presented in this article are those of the authors and do not necessarily reflect those of Worksafe Australia. ⊥ Carnegie Mellon University. # Brookhaven National Laboratory. X Abstract published in Advance ACS Abstracts, April 1, 1997. 1Abbreviations: EXAFS, extended X-ray absorption fine structure; mampa, 5,6-(4,5-dichlorobenzo)-3,8,11,13-tetraoxo-2,2,9,9-tetramethyl12,12-diethyl-1,4,7,10-tetraazacyclotridecanato(4-); NSLS, National Synchrotron Light Source; PIXE, proton-induced X-ray emission; XANES, X-ray absorption near-edge structure; XAS, X-ray absorption spectroscopy. 2C. T. Dillon, P. A. Lay, A. M. Bonin, M. Cholewa, G. J. F. Legge, T. J. Collins, and K. L. Kostka (1996), submitted for publication.
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Cr(III) is bound to intracellular biomolecules after cultured cells are exposed to Cr(VI) (18-20), which immobilizes the Cr and helps to concentrate Cr in the cells. However, there have been no direct determinations of the main oxidation state of Cr in intact cells exposed to carcinogenic Cr(VI) complexes. EPR spectroscopy has been used to monitor the buildup of intracellular Cr(V) and Cr(III) in V79 cells exposed to Cr(VI) (20), but since Cr(VI) is EPR-silent, it is not possible to determine whether Cr(VI) or Cr(III) is the major oxidation state at any given point in the exposure. We report here the first direct determinations of the dominant oxidation state of Cr within Cr(VI)- and Cr(V)-treated mammalian lung cells.
Experimental Procedures Chinese hamster V79 cells were treated, washed, and freezedried according to the methods reported previously for PIXE microprobe experiments (11).2 This involved treatment of the cells with 0.5 µmol of Cr per dish for 4 h. The Cr(VI) and Cr(V) complexes studied were Na2Cr2O7‚2H2O and Li[CrO(mampa)] [mampa ) 5,6-(4,5-dichlorobenzo)-3,8,11,13-tetraoxo-2,2,9,9tetramethyl-12,12-diethyl-1,4,7,10-tetraazacyclotridecanato(4-)] (21), respectively. The microprobe (200-µm diameter) X-ray absorption near-edge structure (XANES) experiments were performed on beamline X26A of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory. The monochromator was a silicon (111) channel-cut crystal mounted on a rotating shaft, which, in turn, was attached to a 230-nm “sine bar”. Other details of the microprobe, calibration, and procedures used in obtaining the spectra are described elsewhere (22). Samples of Na2CrO4‚4H2O and Cr2O3 (1% w/w mixtures in NaCl) were used as Cr(VI) and Cr(III) standards. The standards were scanned between 7.3 and 8.6 keV in 3-eV steps while the cells were scanned between 7.3 and 8.3 keV in 0.5-eV steps. The integration time at each point for the
© 1997 American Chemical Society
534 Chem. Res. Toxicol., Vol. 10, No. 5, 1997
Figure 1. Microprobe XANES spectra obtained from (a) Na2CrO4‚4H2O as a Cr(VI) standard; (b) Cr2O3 as a Cr(III) standard; (c) V79 cells treated with 0.5 µmol/plate of Cr(VI) for 4 h; and (d) V79 cells treated with 0.5 µmol/plate of [CrO(mampa)]- for 4 h. standards was 10 s, while those for the Cr(VI)- and Cr(V)-treated cells were 40 s and 20 s, respectively. Caution: Cr(VI) compounds are carcinogenic (1), and chromium(V) compounds are genotoxic and potentially carcinogenic (7, 10). Appropriate care should be taken to avoid skin contact and inhalation of dust.
Results and Discussion The K-edge XAS spectrum of [CrO4]2- is characterized by an intense pre-edge peak due to the symmetryforbidden 1sf3d transition (Figure 1a). This transition achieves considerable intensity from mixing of p and d orbitals due to the tetrahedral symmetry of the complexes and the strong π bonding involved in the CrdO bonds. XANES and extended X-ray absorption fine structure (EXAFS) analyses of five-coordinate oxoCr(V) species with a large number of ligand sets have also been performed.3 All are characterized by a moderate intensity 1sf3d transition for the same reasons as outlined above. Neither the symmetry nor the π-bonding effects are as important for the octahedral Cr(III) complexes, and, hence, there are only very weak pre-edge features in the spectrum (Figure 1b). Apart from the pre-edge features, the edge for the Cr(III) standard is approximately 5 eV lower in energy compared with the Cr(VI) complex (Figure 1a,b). The difference is somewhat less for a large range of Cr(V) and Cr(III) complexes, but is still significant. No Cr K-edge XAS edges were observed above the background noise for control cells that had not been treated with Cr compounds or those that were treated with Cr(III) complexes. For the cells treated with 0.5 µmol of Cr(VI) per dish for 4 h, it is clear that all of the Cr(VI) (>90%) had been reduced to Cr(III), as evidenced by the shift in the edge and the disappearance of the preedge peak typical of Cr(VI) (Figure 1c). The XANES region is somewhat different, however, because of the different coordination environment(s) of Cr(III) in the cell compared with the standard. The Cr(V)-treated cells (Figure 1d) absorbed X-rays with a similar edge energy as the Cr(III) standard and the Cr(VI)-treated cells. They also show the disappearance of the typical moderateintensity pre-edge peak of Cr(V) (typically ∼20% of the 3G. Barr-David, R. Codd, H. C. Freeman, P. A. Lay, T. Maschmeyer, A. F. Masters, and L. Zhang, unpublished results.
Communications
intensity of the maximum peak at the edge)3 and produced XANES spectra typical of non-thiol-containing Cr(III)-peptide and amino acid complexes.4 The height of the pre-edge peak in the Cr(V)-treated cell is ∼2% of the most intense peak, which is typical for Cr(III).4 Although we cannot preclude the possibility of up to 20% of Cr(V) remaining in the cell from the intensity of the pre-edge peak, the combination of the intensity of the preedge and the edge shape and position indicated >90% reduction to Cr(III). The shapes of the edge and XANES structures in the spectra of the Cr(V)-treated cells are different from those obtained from the Cr(VI)-treated cells. This shows that the nature of the Cr(III) products is different following treatment of the cells with different Cr complexes. This is expected, in the instance of treatment with the [CrO(mampa)]- complex compared with other complexes, since the macrocyclic ligand in the Cr(V) complex is inert to substitution in the biological medium (21, 23).2 The results reported here show that microprobe XAS experiments can be used to determine the intracellular chromium oxidation states within intact mammalian lung cells exposed to carcinogenic and mutagenic Cr complexes. This has considerable potential for examining the time course for intracellular reduction of Cr(VI) and Cr(V) complexes and, hence, will provide further insights into understanding the metabolism of such carcinogenic complexes.
Acknowledgment. We thank Mr. Tony Romeo for technical support. We are grateful for beamtime at Brookhaven National Laboratory’s NSLS, and financial support from the Australian National Science and Technology Organisation, Access to Major Facilities Program. We are also grateful for support from the following sources: P.A.L., the Australian Research Council (ARC) and the National Health and Medical Research Council; G.J.F.L., ARC; and T.J.C., NSF Grant CHE-9319505.
References (1) IARC (1990) IARC Monographs on the Evaluation of Carcinogenic Risk to Humans: Chromium, Nickel and Welding, Vol. 49, pp 49508, International Agency for Research on Cancer, Lyon. (2) Nriagu, J. O., and Nieboer, E., Eds. (1988) Chromium in the Natural and Human Environments, Wiley, New York. (3) Langård, S., Ed. (1982) Biological and Environmental Aspects of Chromium, Elsevier Biomedical, Amsterdam. (4) Burrows, D., Ed. (1983) Chromium: Metabolism and Toxicity, CRC, Boca Raton. (5) Cohen, M. D., Kargacin, B., Klein, C. B., and Costa, M. (1993) Mechanisms of chromium carcinogenicity and toxicity. Crit. Rev. Toxicol. 23, 225-281. (6) De Flora, S., Bagnasco, M., Serra, D., and Zanacchi, P. (1990) Genotoxicity of chromium compounds. A review. Mutat. Res. 238, 99-172. (7) Farrell, R. P., Judd, R. J., Lay, P. A., Dixon, N. E., Baker, R. S. U., and Bonin, A. M. (1989) Chromium(V)-induced cleavage of DNA: are chromium(V) complexes the active carcinogens in chromium(VI)-induced cancers? Chem. Res. Toxicol. 2, 227-229. (8) Kortenkamp, A., Ozolins, Z., Beyersmann, D., and O’Brien, P. (1989) Generation of PM2 DNA breaks in the course of reduction of chromium(VI) by glutathione. Mutat. Res. 216, 19-26. (9) Barr-David, G., Hambley, T. W., Irwin, J. A., Judd, R. J., Lay, P. A., Martin, B. D., Bramley, R., Dixon, N. E., Hendry, P., Ji, J.-Y., Baker, R. S. U., and Bonin, A. M. (1992) Suppression by vanadium(IV) of chromium(V)-mediated DNA cleavage and chromium(VI/V)-induced mutagenesis. Synthesis and crystal structure of the vanadium(IV) complex (NH4)[V(O){HOC(Et)2COO}{OC(Et)2COO}]. Inorg. Chem. 31, 4906-4908. (10) Dillon, C. T., Lay, P. A., Bonin, A. M., Dixon, N. E., Collins, T. J., and Kostka, K. L. (1993) In vitro DNA damage and mutations 4H.
Headlam, P. A. Lay, and A. F. Masters, unpublished results.
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(11)
(12) (13) (14)
(15) (16)
induced by a macrocyclic tetraamide chromium(V) complex: implications for the role of Cr(V) peptide complexes in chromiuminduced cancers. Carcinogenesis 14, 1875-1888. Cholewa, M., Turnbull, I. F., Legge, G. J. F., Weigold, H., Marcuccio, S. M., Holan, G., Tomlinson, E., Wright, P. J., Dillon, C. T., Lay, P. A., and Bonin, A. M. (1995) Investigation of the uptake of drugs, carcinogens and mutagens by individual mammalian cells using a scanning proton microprobe. Nucl. Instrum. Methods Phys. Res., Sect. B B104, 317-323. Gray, S. J., and Sterling, K. (1950) The tagging of red cells and plasma proteins with radioactive chromium. J. Clin. Invest. 29, 1604-1613. Debetto, P., Arslan, P., Antolini, M., and Luciani, S. (1988) Uptake of chromate by rat thymocytes and role of glutathione in its cytoplasmic reduction. Xenobiotica 18, 657-664. Popper, H. H., Grygar, E., Ingolic, E., and Wawschinek, O. (1993) Cytotoxicity of chromium-III and -VI compounds. I. In vitro studies using different cell culture systems. Inhalation Toxicol. 5, 345-369. Kortenkamp, A., Beyersmann, D., and O’Brien, P. (1987) Uptake of chromium(III) complexes by erythrocytes. Toxicol. Environ. Chem. 14, 23-32. Debetto, P., Lazzarini, A., Tomasi, A., Beltrame, M., and Arslan, P. (1986) Chromate uptake and oxidation state of intracellular chromium in rat thymocytes. Cell Biol. Int. Rep. 10, 214.
Chem. Res. Toxicol., Vol. 10, No. 5, 1997 535 (17) Connett, P. H., and Wetterhahn, K. E. (1983) Metabolism of the carcinogen chromate by cellular constituents. Struct. Bonding (Berlin) 54, 93-124. (18) Arslan, P., Beltrame, M., and Tomasi, A. (1987) Intracellular chromium reduction. Biochim. Biophys. Acta 931, 10-15. (19) Bryson, W. G., and Goodall, C. M. (1981) Improved spectrophotometric determination of chromium in animal tissue digests with diphenylcarbazide. Anal. Chim. Acta 124, 391-401. (20) Sugiyama, M. (1994) Role of paramagnetic chromium in chromium(VI)-induced damage in cultured mammalian cells. Environ. Health Persp. 102 (Suppl. 3), 31-33. (21) Collins, T. J., Slebodnick, C., and Uffelman, E. S. (1990) Chromium(V)-oxo complexes of macrocyclic tetraamido-N ligands tailored for highly oxidized middle transition metal complexes: a new 18O-labeling reagent and a structure with four nonplanar amides. Inorg. Chem. 29, 3433-3436. (22) Bajt, S., Clark, S. B., Sutton, S. R., Rivers, M. L., and Smith, J. V. (1993) Synchrotron X-ray microprobe determination of chromate content using X-ray absorption near-edge structure. Anal. Chem. 65, 1800-1804. (23) Dillon, C. T. (1995) The role of Cr(V) complexes in chromiuminduced genotoxicities. Ph.D. Thesis, University of Sydney.
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