Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2023 May 1.
Published in final edited form as: Toxicol Appl Pharmacol. 2022 Mar 27;442:115993. doi: 10.1016/j.taap.2022.115993

Expression of Arylamine N-Acetyltransferase 2 Activity in Immortalized Human Bronchial Epithelial Cells

James TF Wise 1, Raúl A Salazar-González 1, Mariam R Habil 1, Mark A Doll 1, David W Hein 1
PMCID: PMC9112076  NIHMSID: NIHMS1795068  PMID: 35353990

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 KM 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: Arylamine N-acetyltransferase 2, immortalized human lung epithelial cells, lung cancer, β-naphthylamine, 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl

INTRODUCTION

Lung cancer is the leading cause of cancer deaths worldwide [1]. Lung cancer is largely viewed as disease in tobacco smokers [1–3]. Tobacco smokes are known to contain carcinogenic agents such as heavy metals, hydrazines, aromatic amines, and alkylanilines [4]. The carcinogenicity of the hydrazines, aromatic amines, and alkylanilines relies on the metabolism by arylamine N-acetyltransferases and interestingly their genotoxicity in human lung epithelial cells is largely understudied.

Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) are polymorphic xenobiotic metabolizing enzymes [5] and there are two isoenzymes found in humans, N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2). Both can perform N-acetylation and O-acetylation of various carcinogens [5] present in tobacco smoke. NATs are a key driver of their genotoxicity. They are encoded by two separate genes located on chromosome 8 and share significant sequence homology at both the mRNA and protein level but exhibit different substrate specificity and tissue distribution. NAT1 has been shown to have a consistent transcript expression in all major organs and tissue types [6–7]. Conversely, NAT2 transcript expression shows highest in the liver, then small intestine, and colon [5]. Single nucleotide polymorphisms (SNPs) in NAT2 result in rapid, intermediate, and slow N-acetylation phenotypes [8–9]. These SNPs result in an altered genotoxicity profile [8].

The role of NAT2 in human lung cancer had largely been inconclusive and mRNA transcript data from human lung tissues and cells has shown that NAT2 has low if any mRNA expression in this tissue [6–7,10–11]. As such, it was thought that only NAT1 is significantly expressed in human lung epithelial cells. Epidemiological studies have reported conflicting conclusions on a relationship of NAT2 phenotype to human lung cancer risk [12–17]. However, a recent meta-analysis reported NAT2 non-rapid (slow and intermediate) phenotypes had a significantly increased risk of lung cancer [18]. Thus, more mechanistic studies are needed in human lung cell culture models to determine if NAT2 has a role in the carcinogenesis and genotoxicity of arylamine carcinogens. These data will enhance understanding of how hydrazines, aromatic amines, alkylanilines, and heterocyclic amines could cause lung cancer in humans

Therefore, the objective of our study was to investigate endogenous NAT2 N-acetylation activity in immortalized human lung epithelial cells. We used two immortalized human lung epithelial cells (BEP2D and HBEC2-KT cells) and carried out experiments using a NAT1 inhibitor (9,10-dihydro-9,10-dioxo-1,2-anthracenediyl diethyl ester [Compound 10]) and selective substrates for NAT1, para-aminobenzoic acid (PABA), and NAT2, sulfamethazine (SMZ) [19]. We then further investigated the N-acetylation of an arylamine carcinogen and the O-acetylation of an N-hydroxy-arylamine carcinogen. Additionally, we also measured the N-acetylation of 4-ABP by BEP2D cells. These data are important in establishing a role of NAT2 in human lung epithelial cells and demonstrating these cell lines metabolize carcinogens found in tobacco smoke.

MATERIALS AND METHODS

Cell Lines, Chemicals, and Reagents

Deoxyguanosine, acetyl-CoA, β-naphthylamine (BNA), 4-aminobiphenyl (4-ABP), para-aminobenzoic acid, gelatin, sulfamethazine, sodium perchlorate, sodium phosphate, methanol, and acetonitrile were purchased from MilliporeSigma (Burlington, MA). Cell culture supplies, trypsin, phosphate buffered saline (PBS), and LHC-8 media were purchased from ThermoFisher (Waltham, MA). N-hydroxy-4-aminobiphenyl and dG-C-8-ABP adduct standard were purchased from Toronto Research Chemicals (North York, ON, Canada) BEP2D cells an E6/E7 HPV immortalized bronchial epithelial airway cell line were generously gifted by Curtis Harris at the NIH and were cultured in LHC-8 [20]. HBEC2-KT cells a cdk24 and hTERT immortalized bronchial epithelial airway cell line were used in collaboration with John P. Wise Sr. at University of Louisville [21].

Cell Culture

BEP2D and HBEC2-KT cells were cultured in LHC-8 [20–21]. Cell culture dishes were precoated with sterile 0.25% gelatin in PBS. Both cell lines were maintained in 37°C with 5% CO2.

Cell Lysate Preparations

Lung cells were lysed in 20 mM sodium phosphate buffer (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, 100 μM phenylmethanesulfonyl fluoride, 1 μg/ml aprotinin, and 1 μM pepstatin A plus 0.2% triton-x-100. Lysates were then placed on a rotator at 4°C for 10 min. Then lysates were centrifuged at 15,000g for 20 min at 4°C and the supernatant was removed, aliquoted and assayed for protein and enzymatic activity as described below.

Measurement of N-Acetyltransferase N-acetylation Activity in Cell Lysates

N-acetylation reactions contained cell lysate (50 μL), acetyl-CoA (1mM) and NAT1 selective substrate PABA (500 μM), NAT2 selective substrate SMZ (500 μM) or varying concentrations of BNA in DMSO. Reactions were conducted in the presence and absence of 25 μM Compound 10 to inhibit NAT1 catalysis. Reactions were conducted for 10 min at 37°C. Substrates and products were separated and quantitated by high performance liquid chromatography (HPLC) as previously described [6].

Measurement of N-acetylation in Cell Culture

Cells were incubated with substrates for 48 h in culture media (at concentrations as noted in the figure legends). After the 48 h incubation, the media was collected in centrifuge tubes with 1/10 volume of 1M acetic acid and then centrifuged at 15,000g for 10 min. For each treatment cells were counted using Beckman Coulter Z1 DUAL (Beckman Coulter, Inc. Brea, CA), and data was used to normalized to number of cells. N-acetylated product in the culture media was separated and quantitated using HPLC (Agilent Technologies 1260 Infinity) for 4-ABP, BNA, PABA, or SMZ as described previously [7]. The HPLC limit of detection was 0.005 nmol [22].

Measurement of N-Acetyltransferase O-Acetylation

N-hydroxy-4-aminobiphenyl (N–OH-ABP) O-acetyltransferase activity was quantitated using reverse phase HPLC as previously described [5]. In brief, reaction mixtures containing 50 μL BEP2D lysate protein, 1 mg/mL deoxyguanosine (dG), 100 μM N–OH-ABP, and 1 mM acetyl-CoA were incubated at 37°C for 10 min. Reactions were then terminated by the addition of 100 μL of water saturated ethyl acetate and centrifuged for 10 min at 13,000g. The organic phase was then removed and evaporated to dryness and resuspended in 100 μL of 10% acetonitrile. The HPLC separation was achieved using 80:20 sodium perchlorate (pH 2.5): acetonitrile over 3 min to 50:50 sodium perchlorate (pH 2.5): acetonitrile. dGC8-ABP adducts were detected by absorbance at 300 nm.

Protein Quantification

Protein concentrations were quantified using Bradford reagent assay, following manufacturer’s protocol (Bio-Rad, Hercules, CA).

Genotyping

Genotypes for NAT1 and NAT2 were determined as previously described [6,23–24]. Analysis was carried out on the Applied Biosystems StepOne™ Plus (Thermofisher, Carlsbad, CA).

“In cell” Western

To measure N-acetyltransferase 1 and 2 protein expression, we used a standardized “in cell” western blot assay and performed the analysis on the LiCor Odyssey as described [25]. The Abcam antibody (ab109114) has been validated as N-acetyltransferase 1 specific and the Abcam antibody (ab194114) has been validated as N-acetyltransferase 2 specific [25]. RedDot™2 Far-Red Nuclear Stain (Biotium, Inc, Fremont, CA) at 0.5x was used for normalization to cell number [25].

Statistical Analysis and Kinetic Data

Statistical tests were performed with a one-way ANOVA followed a multiple comparison for >2 treatments. For 2 or less treatment statistical tests were performed using a student’s t-test. Results were considered significant at P<0.05 and were performed using Prism 9 software (GraphPad Software, La Jolla, CA, USA). Kinetic data were fit to the Michaelis–Menten equation and apparent kinetic parameters were also determined using GraphPad Prism.

RESULTS

NAT1 and NAT2 Genotypes

BEP2D cells have a NAT1 and NAT2 genotype of NAT1*4/*10 and NAT2*4/*5B and HBEC2-KT cells had NAT1 and NAT2 genotype of NAT1*4/*10 and NAT2*4/*6A respectively.

NAT1 and NAT2 Protein Expression and Activity

Both NAT1 and NAT2 protein were expressed in BEP2D and HBEC2-KT human epithelial cells (figure 1). N-acetylation of both PABA and SMZ were dose-dependent in the BEP2D and HBEC2-KT cells (figure 2).

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

Figure 1.

Open in a new tab

(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.

Figure 2.

Open in a new tab

(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.

Measurement of N-Acetyltransferase Activity with and without NAT1 inhibitor

N-acetyltransferase activity in the human lung epithelial cell lysate was measured in the presence and absence of the NAT1 inhibitor Compound 10. For BEP2D cells, the PABA N-acetyltransferase activity was 0.75 ± 0.46 nmol/min/mg protein in the absence of Compound 10 but was undetectable in the presence of 25 μM NAT1 inhibitor (Figure 3A). BEP2D cells exhibited NAT2 N-acetyltransferase activity towards SMZ of 2.68 ± 0.8 and 2.54 ± 0.34 nmol/min/mg protein in the presence of 0 and 25 μM NAT1 inhibitor respectively (figure 3B).

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

Figure 3.

Open in a new tab

(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.

We observed similar results for compound 10 in HBEC2-KT cells (figure 3C–D). PABA N-acetyltransferase activity was 0.22 ± 0.11 nmol/min/mg protein but was undetectable in the presence of 25 μM NAT1 inhibitor. HBEC2-KT SMZ N-acetyltransferase activities were 3.61 ± 1.58 and 3.15 ± 1.07 nmol/min/mg protein in the presence of 0 and 25 μM NAT1 inhibitor respectively. After, these initial experiments, we chose to only focus on the use of BEP2D cells, as this is our preferred model for future studies. BEP2D cells exhibited dose-dependent BNA N-acetyltransferase activities in cell lysates were reduced in the presence of 25 μM NAT1 inhibitor Compound 10 (figure 4A). BEP2D cell lysate also catalyzed the O-acetylation of N-hydroxy-4-ABP and this activity also was reduced in the presence of 25 μM NAT1 inhibitor Compound 10 (figure 5). These data indicate a role for NAT2 in the O-acetylation of N-hydroxy-4-ABP. BEP2D cells showed dose-dependent N-acetylation of 4-ABP with an apparent KM of 24.4 ± 5.1 μM (figure 6).

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

Figure 4.

Open in a new tab

(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) Vmax 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.

Figure 5.

Open in a new tab

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.

Figure 6.

Open in a new tab

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 KM is 24.4 ± 5.1 μM. Data represent Mean ± SEM of at least 3 experiments.

DISCUSSION

Tobacco smoke and environmental factors are recognized as the main causes of lung cancer in humans [1–4]. It is important to further develop the cell culture models for the genotoxicity of inhalation carcinogens. Tobacco smoke contains carcinogenic arylamines such as BNA and 4-ABP [4]. The carcinogenicity of carcinogenic arylamines is dependent on their metabolism and subsequent genotoxicity and this process is understudied in human lung epithelial cells and warrants investigation.

Prior to this study, it was largely believed that NAT2 activity in humans is largely distributed only in the liver and GI tract, while NAT1 was found in most human tissues and cells [6–7,10–11]. NAT2 mRNA and protein levels have been investigated in human lung epithelial cells and tissues but the data has largely been inclusive or negative [4,10]. However, with the recent meta-analysis on NAT2 allele variant and lung cancer [18], we investigated whether immortalized human lung epithelial cells express NAT2. Additionally, given the high expression of NAT1, we incorporated a recently identified NAT1 inhibitor (Compound 10) to minimize or eliminate catalysis by NAT1. Compound 10 is a NAT1 specific inhibitor that is non-competitive and reversible [19]. Previously, it was shown that the in vitro (cell lysate) IC50 for Compound 10 was reported to be 0.75 ± 0.2 and 82.2 ± 12.9 μM for human NAT1 and NAT2 recombinantly expressed in yeast, respectively. The IC50 for NAT1 was 100 times lower than NAT2 [19]. Our results revealed that N-acetylation of SMZ was unaffected by this NAT1 inhibitor, while PABA N-acetylation was abolished and BNA N-acetylation and N-OH-ABP O-acetylation were reduced consistent with expression of human NAT2 in immortalized human lung epithelial cells.

These data represent for the first time that NAT2 activity has been reported in human lung cell lines. It is important to note that the 4-ABP and SMZ N-acetylation rates and NAT2 protein expression level were dramatically lower than what was previously reported in cryopreserved human hepatocytes [25–26]. BEP2D cells exhibited an apparent NAT2 kM for 4-ABP of 24.4 ± 5.1 μM which is consistent with the 4-ABP apparent Km of 25.5 ± 3.3 μM reported for human recombinant NAT2 expressed in bacteria [27] and 23.7 ± 4.9 μM reported for human recombinant NAT2 expressed in yeast [28]. Some studies have investigated the genotoxicity of aromatic amines in lung carcinoma cell lines [29–30]. However, given that these studies were done with cancer cells it is hard to infer what this means for primary lung cells or immortalized lung cells. Our future investigation will look investigate the genotoxicity endpoints for the exposure of inhaled carcinogens that rely on NAT2 metabolism [28]. Our data demonstrate that NAT2 activity is expressed in immortalized human lung epithelial cells and combined with the recent report of lung cancer incidence with NAT2 phenotype [18] suggest that further investigations into the role of NAT2 in the human lung and in human lung cancers is warranted. Future studies are aimed at investigating a mechanistic role for NAT2 in human lung following exposure to carcinogens. Also, we will investigate NAT2 activities in primary lung cultures and tissues, as the data presented here are in immortalized lung cells. Nevertheless, these data demonstrate the utility of this inhalation model for NAT2 substrates.

HIGHLIGHTS.

  • Immortalized human lung epithelial cells express N-acetyltransferase 2 protein.

  • Immortalized human lung epithelial cells express N-acetyltransferase 2 activity.

  • Immortalized human lung epithelial cells express O-acetylation activity catalyzed by N-acetyltransferases 1 and 2.

ACKNOWLEDGEMENTS

This work is supported by NIEHS T32 training grant [T32ES011564], University of Louisville Superfund Research program grant [P42ES023716] and NIEHS P30 center grant [P30ES030283]. We thank Dr. Curtis Harris at the NIH for the gift of the BEP2D cells and Dr. John Wise Sr. at the University of Louisville for the use of the HBEC2-KT cells.

Footnotes

Author credit statement

James TF Wise carried out the majority of experiments and wrote the manuscript with guidance and financial support of the Principal Investigator, David W. Hein. Raúl A. Salazar-González and Mark A. Doll both assisted with experimentation and aided editing the manuscript drafts. Mariam R. Habil helped with a literature review towards formulating the discussion, along with editing the manuscript drafts.

The authors declare they have no actual or potential competing financial interests.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REFERENCES

  • 1.Thandra KC, Barsouk A, Saginala K, Aluru JS, Barsouk A. (2021) Epidemiology of lung cancer. Contemp Oncol (Pozn). 25(1):45–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.National Center for Health Statistics (2013) Deaths: Final Data for 2010. National Vital Stat Rep. 61:1–17. [Google Scholar]
  • 3.American Cancer Society (2014) Cancer Facts & Figures 2014. Atlanta: American Cancer Society. [Google Scholar]
  • 4.Centers for Disease Control and Prevention (US); National Center for Chronic Disease Prevention and Health Promotion (US); Office on Smoking and Health (US). How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease: A Report of the Surgeon General. Atlanta (GA): Centers for Disease Control and Prevention (US); 2010. 3, Chemistry and Toxicology of Cigarette Smoke and Biomarkers of Exposure and Harm. Available from: https://www.ncbi.nlm.nih.gov/books/NBK53014/ [Google Scholar]
  • 5.Hein DW, Doll MA, Nerland DE, Fretland AJ. (2006) Tissue distribution of N-acetyltransferase 1 and 2 catalyzing the N-acetylation of 4-aminobiphenyl and O-acetylation of N-hydroxy-4-aminobiphenyl in the congenic rapid and slow acetylator Syrian hamster. Mol Carcinog. 45(4):230–238. [DOI] [PubMed] [Google Scholar]
  • 6.Doll MA, Zang Y, Moeller T, Hein DW. (2010) Codominant expression of N-acetylation and O-acetylation activities catalyzed by N-acetyltransferase 2 in human hepatocytes. J Pharmacol Exp Ther. 334(2):540–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Husain A, Zhang X, Doll MA, States JC, Barker DF, Hein DW. (2007) Identification of N-acetyltransferase 2 (NAT2) transcription start sites and quantitation of NAT2-specific mRNA in human tissues. Drug Metab Dispos. 35(5):721–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Hein DW. (2002) Molecular genetics and function of NAT1 and NAT2: role in aromatic amine metabolism and carcinogenesis. Mutat Res. 506–507:65–77. [Google Scholar]
  • 9.McDonagh EM, Boukouvala S, Aklillu E, Hein DW, Altman RB, Klein TE. (2014) PharmGKB summary: very important pharmacogene information for N-acetyltransferase 2. Pharmacogenet Genomics. 24(8):409–25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Damiani LA, Yingling CM, Leng S, Romo PE, Nakamura J, Belinsky SA. (2008). Carcinogen-induced gene promoter hypermethylation is mediated by DNMT1 and causal for transformation of immortalized bronchial epithelial cells. Cancer Res. 68(21):9005–14. [DOI] [PubMed] [Google Scholar]
  • 11.Dairou J, Petit E, Ragunathan N, Baeza-Squiban A, Marano F, Dupret JM, Rodrigues-Lima F. (2009) Arylamine N-acetyltransferase activity in bronchial epithelial cells and its inhibition by cellular oxidants. Toxicol Appl Pharmacol. 236(3):366–71. [DOI] [PubMed] [Google Scholar]
  • 12.Wikman H, Thiel S, Jäger B, Schmezer P, Spiegelhalder B, Edler L, Dienemann H, Kayser K, Schulz V, Drings P, Bartsch H. (2001) Relevance of N-acetyltransferase 1 and 2 (NAT1, NAT2) genetic polymorphisms in non-small cell lung cancer susceptibility. Pharmacogenetics and Genomics. 11(2):157–68. [Google Scholar]
  • 13.Liu C, Cui W, Cong L, Wang L, Ruan X, Jia J, Liu Y, Jia X, Zhang X. (2015) Association Between NAT2 Polymorphisms and Lung Cancer Susceptibility. Medicine (Baltimore). 94(49):e1947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Cui D, Wang Z, Zhao E, Ma J, Lu W. (2011) NAT2 polymorphism and lung cancer risk: a meta-analysis. Lung Cancer. 73(2):153–7. [DOI] [PubMed] [Google Scholar]
  • 15.Agúndez JA. (2008) Polymorphisms of human N-acetyltransferases and cancer risk. Curr Drug Metab. 9(6):520–31. [DOI] [PubMed] [Google Scholar]
  • 16.Bouchardy C, Mitrunen K, Wikman H, Husgafvel-Pursiainen K, Dayer P, Benhamou S, Hirvonen A. (1998) N-acetyltransferase NAT1 and NAT2 genotypes and lung cancer risk. Pharmacogenetics. 8(4):291–8. [DOI] [PubMed] [Google Scholar]
  • 17.Hein DW, Doll MA, Fretland AJ, Leff MA, Webb SJ, Xiao GH, Devanaboyina US, Nangju NA, Feng Y. (2000) Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms. Cancer Epidemiol Biomarkers Prev.9(1):29–42. [PubMed] [Google Scholar]
  • 18.Zhu K, Xu A, Xia W, Li P, Zhang B, Jiang H, Zhou S, Wang R. (2021) Association Between NAT2 Polymorphism and Lung Cancer Risk: A Systematic Review and Meta-Analysis. Front Oncol. 11:567762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Leggett CS, Doll MA, Salazar-González RA, Habil MR, Trent JO, Hein DW. (2022) Identification and characterization of potent, selective, and efficacious inhibitors of human arylamine N-acetyltransferase 1. Arch Toxicol 96 (2): 511–524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Willey JC, Broussoud A, Sleemi A, Bennett WP, Cerutti P, Harris CC. (1991) Immortalization of normal human bronchial epithelial cells by human papillomaviruses 16 or 18. Cancer Res. 51(19):5370–7. [PubMed] [Google Scholar]
  • 21.Ramirez RD, Sheridan S, Girard L, Sato M, Kim Y, Pollack J, Peyton M, Zou Y, Kurie JM, DiMaio JM, Milchgrub S. (2004) Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer research. 64(24):9027–34. [DOI] [PubMed] [Google Scholar]
  • 22.Stepp MW, Salazar-González RA, Hong KU, Doll MA, Hein DW. (2019) N-Acetyltransferase 1 Knockout Elevates Acetyl Coenzyme A Levels and Reduces Anchorage-Independent Growth in Human Breast Cancer Cell Lines. J Oncol. 2019:3860426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Doll MA, Hein DW. Comprehensive human NAT2 genotype method using single nucleotide polymorphism-specific polymerase chain reaction primers and fluorogenic probes. (2001) Anal Biochem. 288(1):106–8. [DOI] [PubMed] [Google Scholar]
  • 24.Doll MA, Hein DW. (2002) Rapid genotype method to distinguish frequent and/or functional polymorphisms in human N-acetyltransferase-1. Anal Biochem. 301(2):328–32. [DOI] [PubMed] [Google Scholar]
  • 25.Salazar-González RA, Doll MA, Hein DW. (2020) Human arylamine N-acetyltransferase 2 genotype-dependent protein expression in cryopreserved human hepatocytes. Sci Rep. 10(1):1–2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Habil MR, Doll MA, Hein DW. (2020) N-acetyltransferase 2 acetylator genotype-dependent N-acetylation of 4-aminobiphenyl in cryopreserved human hepatocytes. Pharmacogenet Genomics. 30(3):61–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Hein DW, Doll MA, Rustan TD, Gray K, Feng Y, Ferguson RJ, Grant DM. (1993) Metabolic activation and deactivation of arylamine carcinogens by recombinant human NAT1 and polymorphic NAT2 acetyltransferases. Carcinogenesis. 14(8):1633–8. [DOI] [PubMed] [Google Scholar]
  • 28.Leggett CS, Doll MA, States JC, Hein DW. (2021) Acetylation of putative arylamine and alkylaniline carcinogens in immortalized human fibroblasts transfected with rapid and slow acetylator N-acetyltransferase 2 haplotypes. Arch Toxicol. 95(1):311–319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Bui LC, Manaa A, Xu X, Duval R, Busi F, Dupret JM, Rodrigues-Lima F, Dairou J. (2013) Acrolein, an α,β-unsaturated aldehyde, irreversibly inhibits the acetylation of aromatic amine xenobiotics by human arylamine N-acetyltransferase 1. Drug Metab Dispos. 41(7):1300–5. [DOI] [PubMed] [Google Scholar]
  • 30.Juricek L, Bui LC, Busi F, Pierre S, Guyot E, Lamouri A, Dupret JM, Barouki R, Coumoul X, Rodrigues-Lima F. (2015) Activation of the aryl hydrocarbon receptor by carcinogenic aromatic amines and modulatory effects of their N-acetylated metabolites. Arch Toxicol. 89(12):2403–12. [DOI] [PubMed] [Google Scholar]

RESOURCES