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J. Biol. Chem., Vol. 283, Issue 18, 11913-11923, May 2, 2008

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Inhibition of the Human Thioredoxin System

A MOLECULAR MECHANISM OF MERCURY TOXICITY^*

Cristina M. L. Carvalho, Eng-Hui Chew¹, Seyed Isaac Hashemy¹, Jun Lu, and Arne Holmgren²

From the I-Med, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal and The Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden

Mercury toxicity mediated by different forms of mercury is a major health problem; however, the molecular mechanisms underlying toxicity remain elusive. We analyzed the effects of mercuric chloride (HgCl₂) and monomethylmercury (MeHg) on the proteins of the mammalian thioredoxin system, thioredoxin reductase (TrxR) and thioredoxin (Trx), and of the glutaredoxin system, glutathione reductase (GR) and glutaredoxin (Grx). HgCl₂ and MeHg inhibited recombinant rat TrxR with IC₅₀ values of 7.2 and 19.7 nM, respectively. Fully reduced human Trx1 bound mercury and lost all five free thiols and activity after incubation with HgCl₂ or MeHg, but only HgCl₂ generated dimers. Mass spectra analysis demonstrated binding of 2.5 mol of Hg²⁺ and 5 mol of MeHg⁺/mol of Trx1 with the very strong Hg²⁺ complexes involving active site and structural disulfides. Inhibition of both TrxR and Trx activity was observed in HeLa and HEK 293 cells treated with HgCl₂ or MeHg. GR was inhibited by HgCl₂ and MeHg in vitro, but no decrease in GR activity was detected in cell extracts treated with mercurials. Human Grx1 showed similar reactivity as Trx1 with both mercurial compounds, with the loss of all free thiols and Grx dimerization in the presence of HgCl₂, but no inhibition of Grx activity was observed in lysates of HeLa cells exposed to mercury. Overall, mercury inhibition was selective toward the thioredoxin system. In particular, the remarkable potency of the mercury compounds to bind to the selenol-thiol in the active site of TrxR should be a major molecular mechanism of mercury toxicity.

Received for publication, December 12, 2007 , and in revised form, February 25, 2008.

^* This work was supported by Swedish Cancer Society Grant 961, Swedish Research Council Medicine Grant 13x-3529, the K. A. Wallenberg Foundation, and the Karolinska Institutet. 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 on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 46-8-52487686; Fax: 46-8-7284716; E-mail: arne.holmgren{at}ki.se.

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