Human arylamine N-acetyltransferase 1: In vitro and intracellular inactivation by nitrosoarene metabolites of toxic and carcinogenic arylamines

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.