Human arylamine N-acetyltransferase 1

In vitro and intracellular inactivation by nitrosoarene metabolites of toxic and carcinogenic arylamines

Li Liu, Carston R Wagner, Patrick E. Hanna

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Arylamines (ArNH2) are common environmental contaminants, some of which are confirmed risk factors for cancer. Biotransformation of the amino group of arylamines involves competing pathways of oxidation and N-acetylation. Nitrosoarenes, which are products of the oxidation pathway, are electrophiles that react with cellular thiols to form sulfinamide adducts. The arylamine N-acetyltransferases, NAT1 and NAT2, catalyze N-acetylation of arylamines and play central roles in their detoxification. We hypothesized that 4-nitrosobiphenyl (4-NO-BP) and 2-nitrosofluorene (2-NO-F), which are nitroso metabolites of arylamines that are readily N-acetylated by NAT1, would be potent inactivators of NAT1 and that nitrosobenzene (NO-B) and 2-nitrosotoluene (2-NO-T), which are nitroso metabolites of arylamines that are less readily acetylated by NAT1, would be less effective inactivators. The second order rate constants for inactivation of NATl by 4-NO-BP and 2-NO-F were 59200 and 34500 M-1 s-1, respectively; the values for NO-B and 2-NO-T were 25 and 23 M-1 -1. Densitometry quantification and comparisons of specific activities with those of homogeneous recombinant NAT1 showed that NATl constitutes approximately 0.002% of cytosolic protein in HeLa cells. Treatment of HeLa cells with 4-NO-BP (2.5 μÌ) for 1 h caused a 40% reduction in NAT1 activity, and 4-NO-BP (10 μ) caused a 50% loss of NATl activity within 30 min without affecting either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or glutathione reductase (GR) activities. 2-NO-F (1 μM) inhibited HeLa cell NAT1 activity by 36% in 1 h, and a 10 ̀Ì concentration of 2-NO-F reduced NATl activity by 70% in 30 min without inhibiting GAPDH or GR. Mass spectrometric analysis of NAT1 from HeLa cells in which NATl was overexpressed showed that treatment of the cells with 4-NO-BP resulted in sulfinamide adduct formation. These results indicated that exposure to low concentrations of nitrosoarenes may lead to a loss of NAT1 activity, thereby compromising a critical detoxification process.

Original languageEnglish (US)
Pages (from-to)2005-2016
Number of pages12
JournalChemical Research in Toxicology
Volume21
Issue number10
DOIs
StatePublished - Oct 1 2008

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Poisons
Metabolites
HeLa Cells
Acetylation
Detoxification
Glyceraldehyde-3-Phosphate Dehydrogenases
Glutathione Reductase
Arylamine N-Acetyltransferase
Oxidation
Densitometry
Biotransformation
Sulfhydryl Compounds
Rate constants
Cells
N-acetyltransferase 1
4-nitrosobiphenyl
In Vitro Techniques
Impurities
2-nitrosofluorene
Neoplasms

Cite this

Human arylamine N-acetyltransferase 1 : In vitro and intracellular inactivation by nitrosoarene metabolites of toxic and carcinogenic arylamines. / Liu, Li; Wagner, Carston R; Hanna, Patrick E.

In: Chemical Research in Toxicology, Vol. 21, No. 10, 01.10.2008, p. 2005-2016.

Research output: Contribution to journalArticle

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abstract = "Arylamines (ArNH2) are common environmental contaminants, some of which are confirmed risk factors for cancer. Biotransformation of the amino group of arylamines involves competing pathways of oxidation and N-acetylation. Nitrosoarenes, which are products of the oxidation pathway, are electrophiles that react with cellular thiols to form sulfinamide adducts. The arylamine N-acetyltransferases, NAT1 and NAT2, catalyze N-acetylation of arylamines and play central roles in their detoxification. We hypothesized that 4-nitrosobiphenyl (4-NO-BP) and 2-nitrosofluorene (2-NO-F), which are nitroso metabolites of arylamines that are readily N-acetylated by NAT1, would be potent inactivators of NAT1 and that nitrosobenzene (NO-B) and 2-nitrosotoluene (2-NO-T), which are nitroso metabolites of arylamines that are less readily acetylated by NAT1, would be less effective inactivators. The second order rate constants for inactivation of NATl by 4-NO-BP and 2-NO-F were 59200 and 34500 M-1 s-1, respectively; the values for NO-B and 2-NO-T were 25 and 23 M-1 -1. Densitometry quantification and comparisons of specific activities with those of homogeneous recombinant NAT1 showed that NATl constitutes approximately 0.002{\%} of cytosolic protein in HeLa cells. Treatment of HeLa cells with 4-NO-BP (2.5 μ{\`I}) for 1 h caused a 40{\%} reduction in NAT1 activity, and 4-NO-BP (10 μ) caused a 50{\%} loss of NATl activity within 30 min without affecting either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or glutathione reductase (GR) activities. 2-NO-F (1 μM) inhibited HeLa cell NAT1 activity by 36{\%} in 1 h, and a 10 ̀{\`I} concentration of 2-NO-F reduced NATl activity by 70{\%} in 30 min without inhibiting GAPDH or GR. Mass spectrometric analysis of NAT1 from HeLa cells in which NATl was overexpressed showed that treatment of the cells with 4-NO-BP resulted in sulfinamide adduct formation. These results indicated that exposure to low concentrations of nitrosoarenes may lead to a loss of NAT1 activity, thereby compromising a critical detoxification process.",
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AB - Arylamines (ArNH2) are common environmental contaminants, some of which are confirmed risk factors for cancer. Biotransformation of the amino group of arylamines involves competing pathways of oxidation and N-acetylation. Nitrosoarenes, which are products of the oxidation pathway, are electrophiles that react with cellular thiols to form sulfinamide adducts. The arylamine N-acetyltransferases, NAT1 and NAT2, catalyze N-acetylation of arylamines and play central roles in their detoxification. We hypothesized that 4-nitrosobiphenyl (4-NO-BP) and 2-nitrosofluorene (2-NO-F), which are nitroso metabolites of arylamines that are readily N-acetylated by NAT1, would be potent inactivators of NAT1 and that nitrosobenzene (NO-B) and 2-nitrosotoluene (2-NO-T), which are nitroso metabolites of arylamines that are less readily acetylated by NAT1, would be less effective inactivators. The second order rate constants for inactivation of NATl by 4-NO-BP and 2-NO-F were 59200 and 34500 M-1 s-1, respectively; the values for NO-B and 2-NO-T were 25 and 23 M-1 -1. Densitometry quantification and comparisons of specific activities with those of homogeneous recombinant NAT1 showed that NATl constitutes approximately 0.002% of cytosolic protein in HeLa cells. Treatment of HeLa cells with 4-NO-BP (2.5 μÌ) for 1 h caused a 40% reduction in NAT1 activity, and 4-NO-BP (10 μ) caused a 50% loss of NATl activity within 30 min without affecting either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or glutathione reductase (GR) activities. 2-NO-F (1 μM) inhibited HeLa cell NAT1 activity by 36% in 1 h, and a 10 ̀Ì concentration of 2-NO-F reduced NATl activity by 70% in 30 min without inhibiting GAPDH or GR. Mass spectrometric analysis of NAT1 from HeLa cells in which NATl was overexpressed showed that treatment of the cells with 4-NO-BP resulted in sulfinamide adduct formation. These results indicated that exposure to low concentrations of nitrosoarenes may lead to a loss of NAT1 activity, thereby compromising a critical detoxification process.

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