Depletion of intracellular ascorbate by the carcinogenic metals nickel and cobalt results in the induction of hypoxic stress

Konstantin Salnikow, Steven P. Donald, Richard K. Bruick, Anatoly Zhitkovich, James M. Phang, Kazimierz S. Kasprzak

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266 Scopus citations


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 HIFα 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 HIFα 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-1α 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.

Original languageEnglish (US)
Pages (from-to)40337-40344
Number of pages8
JournalJournal of Biological Chemistry
Issue number39
StatePublished - Sep 24 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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