Expression of arylamine N-acetyltransferase 2 activity in immortalized human bronchial epithelial cells

Abstract

Lung cancer is the leading cause of cancer deaths in the United States with high incidence in tobacco smokers. Arylamine N-acetyltransferase 2 (NAT2) is a xenobiotic enzyme that catalyzes both N- and O-acetylation of carcinogens present in tobacco smoke and contributes towards the genotoxicity of these carcinogens. NAT2 allelic variants result in slow, intermediate, and rapid acetylation phenotypes. A recent meta-analysis reported NAT2 non-rapid (slow and intermediate) phenotypes had a significantly increased risk of lung cancer. NAT2 activity in humans is thought to be restricted to liver and gastrointestinal tract, and no studies to our knowledge have reported the expression of NAT2 activity in immortalized human lung epithelial cells. Given the importance of NAT2 in cancer and inhalation of various carcinogens directly into the lungs, we investigated NAT2 activity in human lung epithelial cells. Both NAT1 and NAT2 protein were detected by "in-cell" Western. Arylamine N-acetyltransferase activity was determined with selective substrates for NAT1 (p-aminobenzoic acid; PABA) and NAT2 (sulfamethazine; SMZ) in the presence and absence of a selective NAT1 inhibitor. PABA N-acetylation (NAT1 activity) in cell protein lysates was abolished in the presence of 25 μM of NAT1 inhibitor whereas SMZ N-acetylation (NAT2) was unaffected. Incubation with the NAT1 inhibitor partially reduced the N-acetylation of β-naphthylamine and the O-acetylation of N-hydroxy-4-aminobiphenyl consistent with catalysis by both NAT1 and NAT2. Immortalized human lung epithelial cells exhibited dose-dependent N-acetylation of 4-ABP with an apparent K_M of 24.4 ± 5.1 μM. These data establish that NAT2 is expressed and functional in immortalized human lung epithelial cells and will help us further our understanding of NAT2 in lung cancer.

Keywords: 4-aminobiphenyl; Arylamine N-acetyltransferase 2; Immortalized human lung epithelial cells; Lung cancer; N-hydroxy-4-aminobiphenyl; β-naphthylamine.

Figure 1.. Expression of NAT2 Protein in Immortalized Human Bronchial Epithelial Cells.

(A) The relative expression of NAT1 and NAT2 in BEP2D. Cells as measured by “in cell” western. (B) The relative expression of NAT1 and NAT2 in HBEC2-KT. Cells as measured by “in cell” western. Data normalized to cell density, expressed as relative fluorescent units (RFU). Data represent the average of 2 experiments.

Figure 2.. Immortalized Human Bronchial Epithelial Cells Exhibit N-Acetyltransferase 1 and 2 Activities.

(A) N-acetylated PABA levels in BEP2D cells after 48 h incubation with 0, 3.9, 7.8, 15.6 62.5, 125, 250, and 500 μM PABA. (B) N-acetylated SMZ levels in BEP2D cells after 48 h incubation with 0, 62.5, 125, 250, and 500 μM SMZ. (C) N-acetylated PABA levels in HBEC2-KT cells after 48 h incubation with 0, 3.9, 7.8 and 15.6 μM PABA. (D) N-acetylated SMZ levels in HBEC2-KT cells after 48 h incubation with 0, 62.5, 125, 250, and 500 μM SMZ. Data represent Mean ± SEM of at least 3 experiments.

Figure 3.. NAT1 Inhibitor (Compound 10) Does Not Affect NAT2 Activity in Cell Lysates.

(A) N-acetylated PABA levels in BEP2D cell lysate after 10 min incubation with 500 μM PABA with or without 25 μM NAT1 inhibitor, Compound 10. (B) N-acetylated SMZ levels in BEP2D cell lysate after 10 min incubation with 500 μM SMZ with or without 25 μM NAT1 inhibitor, Compound 10. (C) N-acetylated PABA levels in HBEC2-KT cell lysate after 10 min incubation with 500 μM PABA with or without 25 μM NAT1 inhibitor, Compound 10. (D) N-acetylated SMZ levels in HBEC2-KT cell lysate after 10 min incubation with 500 μM SMZ with or without 25 μM NAT1 inhibitor, Compound 10. Data represent the average of at least 3 experiments ± SEM.

Figure 4.. N-Acetylation of β-naphthylamine in Immortalized Human Bronchial Epithelial Cell Lysate

(A) N-acetylated β-naphthylamine levels in BEP2D cell lysate after incubation with 0, 11, 33, 100, 300, and 900 μM BNA with or without 25 μM NAT1 Inhibitor, Compound 10. (B) V_max of β-naphthylamine in BEP2D cell lysate with or without 25 μM NAT inhibitor, Compound 10. Data represent Mean ± SEM of at least 3 experiments.

Figure 5.. O-Acetylation of N-hydroxy-4-Aminobiphenyl in Immortalized Human Bronchial Epithelial Cell Lysate.

O-Acetylation of N-hydroxy-4-aminobiphenyl (N-OH-ABP) in BEP2D cell lysate after 10 min incubation with 150 and 300 μM N-OH-ABP with or without 25 μM NAT1 inhibitor, Compound 10. No AcoA indicates a reaction with no acetyl-CoA (negative control). Data represent Mean ± SEM of at least 3 experiments.

Figure 6.. N-Acetylation of 4-aminobiphenyl in Immortalized Human Bronchial Epithelial Cells.

N-acetylated 4-aminobiphenyl levels in BEP2D cells after 48 h incubation with 0, 1, 5, 10, and 50 μM 4-ABP. The 4-ABP apparent K_M is 24.4 ± 5.1 μM. Data represent Mean ± SEM of at least 3 experiments.