Chem. Res. Toxicol. 2010, 23, 1647–1649
1647
Consistent Chemical Form of Cd in Liver and Kidney Tissues in Rats Dosed with a Range of Cd Treatments: XAS of Intact Tissues Violet Diacomanolis,† Jack C. Ng,† Ross Sadler,‡ Masaharu Nomura,§ Barry N. Noller,⊥ and Hugh H. Harris*,# The UniVersity of Queensland, National Research Centre for EnVironmental Toxicology-EnTox, Coopers Plains, QLD 4008, Australia, School of Public Health, Griffith UniVersity, Meadowbrook, QLD 4131, Australia, Institute of Materials Structure Science, Photon Factory, Tsukuba, Japan, The UniVersity of Queensland, The Centre for Mined Land Rehabilitation, St. Lucia, QLD 4072, Australia, and The School of Chemistry and Physics, The UniVersity of Adelaide, SA 5005, Australia ReceiVed September 26, 2010
X-ray absorption spectroscopy of frozen intact tissues shows that in rats exposed to a range of treatments involving cadmium, alone or in combination with other metal ions, the coordination environment of cadmium is consistent in both the liver and kidney. Comparison of the spectra from the rat tissues to biologically relevant model compounds indicates that the vast majority of the cadmium is bound to metallothionein in these tissues. Anthropogenic or natural contamination of soil and water with cadmium results in bioaccumulation by terrestrial and aquatic plants and animals, which may, in turn, lead to human uptake by a number of routes. Deleterious public health outcomes from low-level chronic exposure to environmental cadmium are now increasingly recognized (1, 2) with target organs identified including the endocrine system (3), liver (4), and kidney (5), as well as noted carcinogenicity (6). The underlying mechanisms for these observed toxic effects in mammals are, however, still poorly understood, presumably due to the low concentrations of Cd involved and the variety of potential biomolecular targets and associated biochemistry. The soft Lewis acid nature of Cd indicates that many of these mechanisms will involve the formation of thiolate-bound species, but the relative importance of small molecules against protein or other large biomolecules bound to Cd2+ species is uncertain. The liver and kidney play key roles in the detoxification of Cd and its excretion (7). Metallothionein loading with Cd occurs in the liver; the mobile loaded protein can then release Cd in the proximal tubule cells of the kidney after reabsorption from the glomerular filtrate. This triggers the expression of metallothionein in the kidney, and presumably, Cd is then bound to the new protein (7). We have assessed, using X-ray absorption near-edge spectroscopy (XANES1), the chemical form of cadmium in intact liver and kidney samples harvested from rats subjected to a range of dosing regimes involving cadmium alone or in combination with As, Zn, and/or Cu. This reveals the average local coordination environment of cadmium accumulated in these organs. * To whom correspondence should be addressed. Tel: +61-8-8303-5060. Fax: +61-8-8303-4358. E-mail:
[email protected]. † National Research Centre for Environmental Toxicology-EnTox, The University of Queensland. ‡ Griffith University. § Photon Factory. ⊥ The Centre for Mined Land Rehabilitation, The University of Queensland. # The University of Adelaide. 1 Abbreviations: EXAFS, extended X-ray absorption fine structure; XANES, X-ray absorption near-edge structure.
Figure 1. Cd K-edge XANES spectra of frozen liver and kidney tissues harvested from rats subjected to a range of Cd exposures. Refer to Table S1 (Supporting Information) for treatment details. Data from traces b, f, g, m, and n are from ref 15.
Cd K-edge XANES spectra for frozen harvested kidney and liver rat tissues are shown in Figure 1. It is clearly evident that all spectra are very similar despite a large variation in treatments presented to animals. Cd K-edge near-edge spectra are diagnostic for small variations in the coordination environment of Cd and can be used to easily distinguish tetragonally thiolate-coordinated
10.1021/tx100333r 2010 American Chemical Society Published on Web 11/01/2010
1648
Chem. Res. Toxicol., Vol. 23, No. 11, 2010
Figure 2. Comparison of the Cd K-edge XANES spectrum (broken lines) of the rat liver (Figure 1, trace g) against those of model compounds: a, solid CdO diluted in BN; b, frozen aqueous solution of Cd2+, Cd(H2O)62+; c, Cd-loaded yeast metallothionein (George, G. N., unpublished work); d, solid CdS diluted in BN; e, frozen aqueous solution of Cd2+ after the addition of excess reduced glutathione at pH 7.4 (George, G. N., unpublished work); f, solid CdSe diluted in BN.
species from octahedrally O/N-coordinated complexes (8) (see Figure 2) and even distinguish one tetragonally thiolatecoordinated species from another, although with less certainty. This indicates that the predominant coordination environment for Cd is essentially identical in the liver and kidney, and is not modulated by dosed Cd species, cotreatment with other elements, administration route (oral vs IP vs IV), or by toxic end-point. The similarity of the spectra shown in Figure 1 is verified by principal component analysis where only one component is identified among the set of spectra (see Table S1 and Figure S1, Supporting Information, for calculated eigenvalues and a plot of eigenvectors). Figure 2 shows a comparison of a representative (Figure 1, trace g) Cd K-edge XANES spectrum of rat tissue compared against a series of model compound spectra. Solid state and aqueous oxygen coordinated Cd2+ compounds display edge spectra with a distinct peak at the top of the rising edge. This distinguishes the spectra of those species from the rat spectrum, which lacks a strong edge peak and is a much closer match for the sulfur and selenium bound model spectra shown in traces c-f. Of these, the CdSe can be eliminated as a match with a large discrepancy appearing well above the absorption edge, at the beginning of the extended X-ray absorption fine structure (EXAFS) region. The spectrum of a Cd2+ complex formed with a large excess of reduced glutathione in water (pH 7.4) was also found to be a poor match for the rat tissue spectrum. In contrast, both solid CdS and Cd-loaded yeast metallothionein display Cd edge spectra (Figure 2, traces c and d) that are extremely similar to that of the rat tissues. Initial examination
Rapid Report
of the difference spectra between these models and the rat tissue spectrum (Figure S2, Supporting Information) suggests that the solid CdS is a better model, and indeed, target transformation of the CdS spectrum against the principal component analysis of the rat spectrum set produces a smaller residual than does the metallothionein spectrum. However, the EXAFS Fourier transform (Figure S3, Supporting Information) of the representative rat sample (Figure 1 trace g) does not show a strong peak in the 4 Å range that might correspond to a Cd-Cd interaction that would be expected in solid CdS (9) recorded at cryogenic temperatures (the Cd-Cd peak is dramatically diminished at room temperature due to an increased Debye-Waller factor 9, 10) and has been observed for larger cadmium thiolate clusters (8). The rat EXAFS is consistent in this respect, and in terms of the determined Cd-S bond length of 2.54 Å (see Supporting Information), with the EXAFS reported for Cd-loaded phytochelatin (8), the yeast metallothionein (EXAFS data not shown), and rabbit liver metallothionein (11). Meanwhile, closer inspection of the difference spectra shown in Figure S2 (Supporting Information) reveals that the discrepancy between the rat tissue spectrum and that of the metallothionein is focused on the rising edge, rather than across the whole energy range. We note that this discrepancy may arise from the fact that the two spectra were recorded at different beamlines, likely with different energy resolutions, and that a higher resolution measurement of the metallothionein sample would lead to the type of discrepancy observed. The yeast metallionein XANES spectrum presented here is essentially identical to those reported for Cd7-βR and Cd4-R rabbit liver metallothionein (12) (which are themselves identical within resolution variations) and hence serves as a suitable model for the mammalian system. While there may be different isoforms of metallothionein expressed in the different organs, the fact that the tissue spectra presented here are invariant suggests that the Cd coordination environment does not vary enough between the isoforms to generate distinct spectral signatures. Taken together, we believe that the XANES and EXAFS results indicate that the Cd in the rat tissues examined here is, most likely, predominantly bound to metallothionein or metallothionein-like clusters. This result is at odds with a previous study involving rats subjected to subcutaneous Cd2+ dose rates an order of magnitude lower than the present study, where significant nonmetallothionein-bound fractions were identified in both the liver and kidney by a Cd-saturation/hemoglobin method applied to tissue homogenates (13). The discrepancy again illustrates the inherent danger of tissue homogenization in modulating the biochemistry of metal ions, in a manner similar to that demonstrated for Cr (14). What is more intriguing and more strongly supported by the evidence presented here is that despite the highly variable nature of the Cd exposures and the effect they have on the rats, the local Cd coordination environment is consistent across the investigated tissues. Acknowledgment. Synchrotron access travel funding for this work was provided by the Australian Synchrotron Research Program, which was funded by the Commonwealth of Australia under the Major National Research Facilities Program. The animal experimental component is funded by ARC-Linkage grant (LP0214185) to B.N.N. and J.C.N. and an APAI scholarship to V.D. The research was also supported by ARC grants to H.H.H. (DP0664706, DP0985807-QEII). We thank the Photon Factory, High Energy Accelerator Research Organization (KEK), Tsukuba, Japan for access to beamline NW10A under
Rapid Report
Chem. Res. Toxicol., Vol. 23, No. 11, 2010 1649
the General Access Program. EnTox is a partnership between Queensland Health and the University of Queensland. Supporting Information Available: Complete experimental section including details of rat treatments; results of principal component analysis of rat tissue Cd K-edge XANES spectra, XANES difference spectra, and EXAFS of a representative rat tissue. This material is available free of charge via the Internet at http://pubs.acs.org.
(8)
(9) (10)
References (1) WHO (1992) EnVironmental Health Criteria 134: Cadmium. International Programme on Chemical Safety, World Health Organisation, Geneva, Switzerland. (2) IARC (1993) Monographs on the EValuation of the Carcinogenic Risk of Chemicals to Humans. Vol 58, Beryllium, Cadmium, Mercury and Exposures in the Glass Manufacturing Industry, IARC Press, Lyon, France. (3) Johnson, M. D., Kenney, N., Stoica, A., Hilakivi-Clarke, L., Singh, B., Chepko, G., Clarke, R., Sholler, P. F., Lirio, A. A., Foss, C., Reiter, R., Trock, B., Paik, S., and Martin, M. B. (2003) Cadmium mimics the in vivo effects of estrogen in the uterus and mammary gland. Nat. Med. 9, 1081–1084. (4) Ye, J. L., Mao, W. P., Wu, A. L., Zhang, N. N., Zhang, C., Yu, Y. J., Zhou, L., and Wei, C. J. (2007) Cadmium-induced apoptosis in human normal liver L-02 cells by acting on mitochondria and regulating Ca2+ signals. EnViron. Toxicol. Phar. 24, 45–54. (5) Zalups, R. K., and Ahmad, S. (2003) Molecular handling of cadmium in transporting epithelia. Toxicol. Appl. Pharmacol. 186, 163–188. (6) Jin, Y. H., Clark, A. B., Slebos, R. J. C., Al-Refai, H., Taylor, J. A., Kunkel, T. A., Resnick, M. A., and Gordenin, D. A. (2003) Cadmium is a mutagen that acts by inhibiting mismatch repair. Nat. Genet. 34, 326–329. (7) Chen, L., Jin, T. Y., Huang, B., Chang, X. L., Lei, L. J., Nordberg, G. F., and Nordberg, M. (2006) Plasma metallothionein antibody and
(11)
(12)
(13) (14)
(15)
cadmium-induced renal dysfunction in an occupational population in China. Toxicol. Sci. 91, 104–112. Pickering, I. J., Prince, R. C., George, G. N., Rauser, W. E., Wickramasinghe, W. A., Watson, A. A., Dameron, C. T., Dance, I. G., Fairlie, D. P., and Salt, D. E. (1999) X-ray absorption spectroscopy of cadmium phytochelatin and model systems. BBA-Protiens Enzymol. 1429, 351–364. Levelut, C., Ramos, A., Petiau, J., and Robino, M. (1991) EXAFS Study of the Local-Structure in CdSxSe1-X Compounds. Mat. Sci. Eng. B 8, 251–263. Hosokawa, H., Fujiwara, H., Murakoshi, K., Wada, Y., Yanagida, S., and Satoh, M. (1996) In-situ EXAFS observation of the surface structure of colloidal CdS nanocrystallites in N, N-dimethylformamide. J. Phys. Chem. 100, 6649–6656. Jiang, D. T., Heald, S. M., Sham, T. K., and Stillman, M. J. (1994) Structures of the cadmium, mercury, and zinc thiolate clusters in metallothionein: XAFS study of Zn-7-Mt, Cd-7-Mt, Hg-7-Mt, and Hg18-Mt formed from rabbit liver metallothionein-2. J. Am. Chem. Soc. 116, 11004–11013. Chan, J., Merrifield, M. E., Soldatov, A. V., and Stillman, M. J. (2005) XAFS spectral analysis of the cadmium coordination geometry in cadmium thiolate clusters in metallothionein. Inorg. Chem. 44, 4923– 4933. Goyer, R. A., Miller, C. R., Zhu, S. Y., and Victery, W. (1989) Nonmetallothionein-bound cadmium in the pathogenesis of cadmium nephrotoxicity in the rat. Toxicol. Appl. Pharmacol. 101, 232–244. Levina, A., Harris, H. H., and Lay, P. A. (2007) X-ray absorption and EPR spectroscopic studies of the biotransformations of chromium(VI) in mammalian cells. Is chromodulin an artifact of isolation methods? J. Am. Chem. Soc. 129, 1065–1075. Diacomanolis, V., Ng, J. C., Sadler, R., Harris, H. H., Nomura, M., and Noller, B. N. (2010) The 10th International Conference on Synchrotron Radiation Instrumentation (Garrett, I. G, R., Nugent, K., and Wilkins, S., Eds.) pp 613-616, American Institute of Physics, College Park, MD.
TX100333R
