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J. Biol. Chem. 279 (39): 40337-40344

© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Depletion of Intracellular Ascorbate by the Carcinogenic Metals Nickel and Cobalt Results in the Induction of Hypoxic Stress*

Konstantin Salnikow{ddagger}§, Steven P. Donald{ddagger}, Richard K. Bruick¶||, Anatoly Zhitkovich**, James M. Phang{ddagger}, , and Kazimierz S. Kasprzak{ddagger}

{ddagger}NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702, the Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, and the **Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912

Abstract: Exposure of cells to carcinogenic compounds of nickel(II) and cobalt(II) causes activation of the HIF-1 transcription factor and up-regulates a battery of hypoxia-inducible genes. However, the mechanism of HIF-1 activation by these metals is not known. It was shown recently that hydroxylation of prolines in the HIF{alpha} subunit of HIF-1 is required for its binding with the von Hippel-Lindau tumor suppressor protein and the subsequent proteasomal destruction. Here we show that responsible prolyl hydroxylases are targets for both nickel(II) and cobalt(II) because degradation of a reporter protein containing the oxygen-dependent degradation domain (Pro-402/564) of HIF{alpha} was abolished in a von Hippel-Lindau-dependent manner in cells exposed to nickel(II) or cobalt(II). The enzymatic activity of prolyl hydroxylases depends on iron as the activating metal, 2-oxoglutarate as a co-substrate, and ascorbic acid as a cofactor. Hydroxylase activity can be impaired by the depletion of any of these factors. We found that exposure of cells to nickel(II) or cobalt(II) did not affect the level of intracellular iron. Instead, nickel(II) or cobalt(II) exposure greatly depleted intracellular ascorbate. Co-exposure of cells to metals and ascorbate resulted in the increase of intracellular ascorbate and reversed both metal-induced stabilization of HIF-1{alpha} and HIF-1-dependent gene transcription. Because ascorbate is essential for maintaining iron in prolyl hydroxylases in the active iron(II) state, we suggest that the observed depletion of ascorbate by nickel(II) or cobalt(II) favors iron oxidation and thus inactivation of the enzyme.

Received for publication March 18, 2004. Revision received May 10, 2004.

* This work was supported in part by Grant ES008786 from NIEHS, National Institutes of Health (to A. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| The Michael L. Rosenberg Scholar in Medical Research and supported by a Burroughs Wellcome Fund Career Award in the Biomedical Sciences and the University of Texas Southwestern President's Research Council.

§ To whom correspondence should be addressed: NCI-Frederick, National Institutes of Health, Bldg. 538, Rm. 205 E, Frederick, MD 21702. Tel.: 301-846-5623; Fax: 301-846-5946; E-mail: salnikow{at}

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