Abstract
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 10(4) 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.
Highlights
Arylamine N-acetyltransferases (NATs) play an important role in the detoxification and metabolic activation of a variety of aromatic xenobiotics, including numerous carcinogens
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
As demonstrated by comparative expression level analyses of cellular NAT1 by immunoprecipitation and Western blotting (Fig. 2, A and B, bottom), inactivation obtained with peroxynitrite and SIN1 was not due to proteolysis or changes in NAT1 protein expression induced by the exposures to these compounds
Summary
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. These cytosolic enzymes are encoded by two separate genes located on 8p22, a chromosomal region commonly deleted in certain human cancers [5,6,7] This raised the possibility that the absence of NATs or their inactivation may contribute to carcinogenesis and/or tumor progression [6, 8, 9]. NATs may be prone to inactivation by oxidative reactions as shown recently for NAT1 [25] and for other reactive cysteine-containing enzymes (26 –29)
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