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J. Biol. Chem., Vol. 279, Issue 9, 7708-7714, February 27, 2004

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Peroxynitrite Irreversibly Inactivates the Human Xenobioticmetabolizing Enzyme Arylamine N-Acetyltransferase 1 (NAT1) in Human Breast Cancer Cells

A CELLULAR AND MECHANISTIC STUDY^*

Julien Dairou¶, Noureddine Atmane||, Fernando Rodrigues-Lima**, and Jean-Marie Dupret**

From the CNRS-Unité Mixte de Recherche 7000, Faculté de Médecine Pitié-Salpêtrière, 75013 Paris, France and Unité de Formation et de Recherche de Biochimie, Université Paris 7-Denis Diderot, 75005 Paris, France

Arylamine N-acetyltransferases (NATs) play an important role in the detoxification and metabolic activation of a variety of aromatic xenobiotics, including numerous carcinogens. Both of the human isoforms, NAT1 and NAT2, display interindividual variations, and associations between NAT genotypes and cancer risk have been established. Contrary to NAT2, NAT1 has a ubiquitous tissue distribution and has been shown to be expressed in cancer cells. Given that the activity of NAT1 depends on a reactive cysteine that can be a target for oxidants, we studied whether peroxynitrite, a highly reactive nitrogen species involved in human carcinogenesis, could inhibit the activity of endogenous NAT1 in MCF7 breast cancer cells. We show here that exposure of MCF7 cells to physiological concentrations of peroxynitrite and to a peroxynitrite generator (3-morpholinosydnonimine N-ethylcarbamide, or SIN1) leads to the irreversible inactivation of NAT1 in cells. Further kinetic and mechanistic analyses using recombinant NAT1 showed that the enzyme is rapidly (k_inact = 5 x 10⁴ M^–1·s^–1) and irreversibly inactivated by peroxynitrite. This inactivation is due to oxidative modification of the catalytic cysteine. We conclude that the reducing cellular environment of MCF7 cells does not sufficiently protect NAT1 from peroxynitrite-dependent inactivation and that only high concentrations of reduced glutathione could significantly protect NAT1. Thus, cellular generation of peroxynitrite may contribute to carcinogenesis and tumor progression by weakening key cellular defense enzymes such as NAT1.

Received for publication, October 20, 2003 , and in revised form, December 11, 2003.

^* This work was supported by the Association pour la Recherche sur le Cancer (ARC). 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.

^¶ Holder of a postdoctoral fellowship from Université Paris 7-Denis Diderot.

^|| Holder of a Ph.D. fellowship from le Ministère de la Jeunesse, de l'Education Nationale et de la Recherche.

^** These authors contributed equally to this paper and are to whom correspondence may be addressed. Tel.: 33-1-53-60-08-03; Fax: 33-1-53-60-08-02; E-mail: jmdupret{at}infobiogen.fr or rlima{at}ext.jussieu.fr.

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