Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that plays an important role in the biotransformation of aromatic drugs and carcinogens. NAT1 activity has long been associated with susceptibility to various cancers. Evidence for a role of NAT1 in malignant progression has also been obtained, particularly for breast and prostate cancer. Cisplatin is widely used in chemotherapy against human cancers, and it is thought to act principally by forming DNA adducts. However, recent studies have suggested that some of the pharmacological and/or toxicological effects of cisplatin may be due to the direct targeting and inhibition of certain cellular enzymes. We show here that the exposure of breast cancer cells, known to express functional NAT1 enzyme, to therapeutically relevant concentrations of cisplatin impairs the catalytic activity of endogenous NAT1. Endogenous NAT1 was also found to be inactivated, in vivo, in the tissues of mice treated with cisplatin. Mechanistic studies with purified human NAT1 indicated that this inhibition resulted from the irreversible formation of a cisplatin adduct with the active-site cysteine residue of the enzyme. Kinetic studies suggested that NAT1 interacts rapidly with cisplatin, with a second-order rate inhibition constant of 700 M Ϫ1 min Ϫ1 . This rate constant is one the highest ever reported for the reaction of cisplatin with a biological macromolecule. Few enzymes have been clearly shown to be inactivated by cisplatin. We provide here molecular and cellular evidence suggesting that NAT1 is one of the targets of cisplatin in cells.
BackgroundCadmium (Cd) is a carcinogenic heavy metal of environmental concern. Exposure to both Cd and carcinogenic organic compounds, such as polycyclic aromatic hydrocarbons or aromatic amines (AAs), is a common environmental problem. Human arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that play a key role in the biotransformation of AA carcinogens. Changes in NAT activity have long been associated with variations in susceptibility to different cancers in relation with exposure to certain AAs.ObjectiveWe explored the possible interactions between Cd and the NAT-dependent biotransformation of carcinogenic AAs.MethodsWe exposed purified enzymes, lung epithelial cells, and mouse models to Cd and subsequently analyzed NAT-dependent metabolism of AAs.ResultsWe found that Cd, at biologically relevant concentrations, impairs the NAT-dependent acetylation of carcinogenic AAs such as 2-aminofluorene (2-AF) in lung epithelial cells. NAT activity was strongly impaired in the tissues of mice exposed to Cd. Accordingly, mice exposed to Cd and 2-AF displayed altered in vivo toxicokinetics with a significant decrease (~ 50%) in acetylated 2-AF in plasma. We found that human NAT1 was rapidly and irreversibly inhibited by Cd [median inhibitory concentration (IC50) ≈ 55 nM; rate inhibition constant (kinact) = 5 × 104 M−1 · sec−1], with results of acetyl coenzyme A (acetyl-CoA) protection assays indicating that Cd-mediated inhibition was due to the reaction of metal with the active-site cysteine residue of the enzyme. We found similar results for human NAT2, although this isoform was less sensitive to inactivation (IC50 ≈ 1 μM; kinact = 1 × 104 M−1 · sec−1).ConclusionsOur data suggest that Cd can alter the metabolism of carcinogenic AAs through the impairment of the NAT-dependent pathway, which may have important toxicological consequences.
Disulfiram has been used for decades to treat alcoholism. Its therapeutic effect is thought to be mediated by the irreversible inhibition of aldehyde dehydrogenase. Recent reports have indicated new therapeutic uses of disulfiram, in particular in human cancers. Although the biochemical mechanisms that underlie these effects remain largely unknown, certain enzymes involved in cancer processes have been reported to be targeted by disulfiram. Arylamine N‐acetyltransferase 1 (NAT1) is a xenobiotic‐metabolizing enzyme that biotransforms aromatic amine drugs and carcinogens. In addition to its role in xenobiotic metabolism, several studies have suggested that NAT1 is involved in other physiological and/or pathological processes, such as folate metabolism or cancer progression. In this report, we provide evidence that human NAT1 is a new enzymatic target of disulfiram. We found that disulfiram at clinically relevant concentrations impairs the activity of endogenous NAT1 in human cancer cells. Further mechanistic and kinetic studies indicated that disulfiram reacts irreversibly with the active site cysteine residue of NAT1, leading to its rapid inhibition (IC50 = 3.3 ± 0.1 μm and ki = 6 × 104 m−1·min−1).
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