Skip to main content

Advertisement

SpringerLink
Log in

Catalytic properties and heat stabilities of novel recombinant human N-acetyltransferase 2 allozymes support existence of genetic heterogeneity within the slow acetylator phenotype

  • Toxicogenomics
  • Published:
Archives of Toxicology Aims and scope Submit manuscript

Abstract

Human N-acetyltransferase 2 (NAT2) catalyzes the N-acetylation of numerous aromatic amine drugs such as sulfamethazine (SMZ) and hydrazine drugs such as isoniazid (INH). NAT2 also catalyzes the N-acetylation of aromatic amine carcinogens such as 2-aminofluorene and the O- and N,O-acetylation of aromatic amine and heterocyclic amine metabolites. Genetic polymorphism in NAT2 modifies drug efficacy and toxicity as well as cancer risk. Acetyltransferase catalytic activities and heat stability associated with six novel NAT2 haplotypes (NAT2*6C, NAT2*14C, NAT2*14D, NAT2*14E, NAT2*17, and NAT2*18) were compared with that of the reference NAT2*4 haplotype following recombinant expression in Escherichia coli. N-acetyltransferase activities towards SMZ and INH were significantly (p < 0.0001) lower when catalyzed by the novel recombinant human NAT2 allozymes compared to NAT2 4. SMZ and INH N-acetyltransferase activities catalyzed by NAT2 14C and NAT2 14D were significantly lower (p < 0.001) than catalyzed by NAT2 6C and NAT2 14E. N-Acetylation catalyzed by recombinant human NAT2 17 was over several hundred-fold lower than by recombinant NAT2 4 precluding measurement of its kinetic or heat inactivation constants. Similar results were observed for the O-acetylation of N-hydroxy-2-aminofluorene and N-hydroxy-2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine and the intramolecular N,O-acetylation of N-hydroxy-N-acetyl-2-aminofluorene. The apparent V max of the novel recombinant NAT2 allozymes NAT2 6C, NAT2 14C, NAT2 14D, and NAT2 14E towards AF, 4-aminobiphenyl (ABP), and 3,2′-dimethyl-4-aminobiphenyl (DMABP) were each significantly (p < 0.001) lower while their apparent K m values did not differ significantly (p > 0.05) from recombinant NAT2 4. The apparent V max catalyzed by NAT2 14C and NAT2 14D were significantly lower (p < 0.05) than the apparent V max catalyzed by NAT2 6C and NAT2 14E towards AF, ABP, and DMABP. Heat inactivation rate constants for recombinant human NAT2 14C, 14D, 14E, and 18 were significantly (p < 0.05) higher than NAT2 4. These results provide further evidence of genetic heterogeneity within the NAT2 slow acetylator phenotype.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Agundez JA, Olivera M, Martinez C, Ladero JM, Benitez J (1996) Identification and prevalence study of 17 allelic variants of the human NAT2 gene in a white population. Pharmacogenetics 6(5):423–428

    Article  CAS  PubMed  Google Scholar 

  • Ambrosone CB, Kropp S, Yang J, Yao S, Shields PG, Chang-Claude J (2008) Cigarette smoking, N-acetyltransferase 2 genotypes, and breast cancer risk: pooled analysis and meta-analysis. Cancer Epidemiol Biomark Prev 17(1):15–26

    Article  CAS  Google Scholar 

  • Baumgartner KB, Schlierf TJ, Yang D, Doll MA, Hein DW (2009) N-acetyltransferase 2 genotype modification of active cigarette smoking on breast cancer risk among hispanic and non-hispanic white women. Toxicol Sci 112(1):211–220

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Conlon MS, Johnson KC, Bewick MA, Lafrenie RM, Donner A (2010) Smoking (active and passive), N-acetyltransferase 2, and risk of breast cancer. Cancer Epidemiol 34(2):142–149

    Article  PubMed  Google Scholar 

  • Deitz AC, Zheng W, Leff MA et al (2000) N-Acetyltransferase-2 genetic polymorphism, well-done meat intake, and breast cancer risk among postmenopausal women. Cancer Epidemiol Biomark Prev 9(9):905–910

    CAS  Google Scholar 

  • Deitz AC, Rothman N, Rebbeck TR et al (2004) Impact of misclassification in genotype-exposure interaction studies: example of N-acetyltransferase 2 (NAT2), smoking, and bladder cancer. Cancer Epidemiol Biomarkers Prev 13(9):1543–1546

    CAS  PubMed  Google Scholar 

  • Doll MA, Hein DW (2017) Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes. Arch Toxicol. doi:10.1007/s00204-017-1988-8

    Google Scholar 

  • 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):540–544

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ferguson RJ, Doll MA, Rustan TD, Gray K, Hein DW (1994) Cloning, expression, and functional-characterization of 2 mutant (Nat2(191) and Nat2(341/803)) and wild-type human polymorphic N-acetyltransferase (Nat2) alleles. Drug Metab Dispos 22(3):371–376

    CAS  PubMed  Google Scholar 

  • Fretland AJ, Doll MA, Gray K, Feng Y, Hein DW (1997) Cloning, sequencing, and recombinant expression of NAT1, NAT2, and NAT3 derived from the C3H/HeJ (rapid) and A/HeJ (slow) acetylator inbred mouse: functional characterization of the activation and deactivation of aromatic amine carcinogens. Toxicol Appl Pharmacol 142(2):360–366

    Article  CAS  PubMed  Google Scholar 

  • Fretland AJ, Leff MA, Doll MA, Hein DW (2001) Functional characterization of human N-acetyltransferase 2 (NAT2) single nucleotide polymorphisms. Pharmacogenetics 11(3):207–215

    Article  CAS  PubMed  Google Scholar 

  • Fu Z, Shrubsole MJ, Li G et al (2012) Using gene–environment interaction analyses to clarify the role of well-done meat and heterocyclic amine exposure in the etiology of colorectal polyps. Am J Clin Nutr 96(5):1119–1128

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fu Z, Shrubsole MJ, Li G et al (2013) Interaction of cigarette smoking and carcinogen-metabolizing polymorphisms in the risk of colorectal polyps. Carcinogenesis 34(4):779–786

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Garcia-Closas M, Malats N, Silverman D et al (2005) NAT2 slow acetylation, GSTM1 null genotype, and risk of bladder cancer: results from the Spanish bladder cancer study and meta-analyses. Lancet 366(9486):649–659

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Golka K, Prior V, Blaszkewicz M, Bolt HM (2002) The enhanced bladder cancer susceptibility of NAT2 slow acetylators towards aromatic amines: a review considering ethnic differences. Toxicol Lett 128(1–3):229–241

    Article  CAS  PubMed  Google Scholar 

  • Grant DM, Morike K, Eichelbaum M, Meyer UA (1990) Acetylation pharmacogenetics. The slow acetylator phenotype is caused by decreased or absent arylamine N-acetyltransferase in human liver. J Clin Investig 85(3):968–972

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grant DM, Hughes NC, Janezic SA et al (1997) Human acetyltransferase polymorphisms. Mut Res 376(1–2):61–70

    Article  CAS  Google Scholar 

  • Hein DW (1988) Acetylator genotype and arylamine-induced carcinogenesis. Biochim Biophys Acta 948(1):37–66

    CAS  PubMed  Google Scholar 

  • Hein DW (2002) Molecular genetics and function of NAT1 and NAT2: role in aromatic amine metabolism and carcinogenesis. Mutat Res 506–507:65–77

    Article  PubMed  Google Scholar 

  • Hein DW (2009) N-Acetyltransferase SNPs: emerging concepts serve as a paradigm for understanding complexities of personalized medicine. Expert Opin Drug Metab Toxicol 5(4):353–366

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hein DW (2017) N-acetyltransferase 2 polymorphism and human urinary bladder and breast cancer risk. In: Sim E, Laurieri N (eds) Arylamine N-acetyltransferases in health and disease. World Scientific Publishing, Singapore (in press)

  • Hein DW, Doll MA, Rustan TD et al (1993) Metabolic activation and deactivation of arylamine carcinogens by recombinant human NAT1 and polymorphic NAT2 acetyltransferases. Carcinogenesis 14(8):1633–1638

    Article  CAS  PubMed  Google Scholar 

  • Hein DW, Ferguson RJ, Doll MA, Rustan TD, Gray K (1994a) Molecular genetics of human polymorphic N-acetyltransferase: enzymatic analysis of 15 recombinant wild-type, mutant, and chimeric NAT2 allozymes. Hum Mol Genet 3(5):729–734

    Article  CAS  PubMed  Google Scholar 

  • Hein DW, Rustan TD, Ferguson RJ, Doll MA, Gray K (1994b) Metabolic activation of aromatic and heterocyclic N-hydroxyarylamines by wild-type and mutant recombinant human NAT1 and NAT2 acetyltransferases. Arch Toxicol 68(2):129–133

    Article  CAS  PubMed  Google Scholar 

  • Hein DW, Doll MA, Rustan TD, Ferguson RJ (1995) Metabolic activation of N-hydroxyarylamines and N-hydroxyarylamides by 16 recombinant human NAT2 allozymes: effects of 7 specific NAT2 nucleic acid substitutions. Cancer Res 55(16):3531–3536

    CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Hickman D, Palamanda JR, Unadkat JD, Sim E (1995) Enzyme kinetic properties of human recombinant arylamine N-acetyltransferase 2 allotypic variants expressed in Escherichia coli. Biochem Pharmacol 50(5):697–703

    Article  CAS  PubMed  Google Scholar 

  • Leff MA, Epstein PN, Doll MA et al (1999) Prostate-specific human N-acetyltransferase 2 (NAT2) expression in the mouse. J Pharmacol Exp Ther 290(1):182–187

    CAS  PubMed  Google Scholar 

  • Lin HJ, Han CY, Lin BK, Hardy S (1994) Ethnic distribution of slow acetylator mutations in the polymorphic N-acetyltransferase (NAT2) gene. Pharmacogenetics 4(3):125–134

    Article  CAS  PubMed  Google Scholar 

  • Martinez C, Agundez JA, Olivera M, Martin R, Ladero JM, Benitez J (1995) Lung cancer and mutations at the polymorphic NAT2 gene locus. Pharmacogenetics 5(4):207–214

    Article  CAS  PubMed  Google Scholar 

  • McDonagh EM, Boukouvala S, Aklillu E, Hein DW, Altman RB, Klein TE (2014) PharmGKB summary: very important pharmacogene information for N-acetyltransferase 2. Pharmacogenet Genom 24(8):409–425

    CAS  Google Scholar 

  • Moore LE, Baris DR, Figueroa JD et al (2011) GSTM1 null and NAT2 slow acetylation genotypes, smoking intensity and bladder cancer risk: results from the New England bladder cancer study and NAT2 meta-analysis. Carcinogenesis 32(2):182–189

    Article  CAS  PubMed  Google Scholar 

  • Moslehi R, Chatterjee N, Church TR et al (2006) Cigarette smoking, N-acetyltransferase genes and the risk of advanced colorectal adenoma. Pharmacogenomics 7(6):819–829

    Article  CAS  PubMed  Google Scholar 

  • Ruiz JD, Martinez C, Anderson K et al (2012) The differential effect of NAT2 variant alleles permits refinement in phenotype inference and identifies a very slow acetylation genotype. PLoS One 7(9):e44629

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74(12):5463–5467

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Selinski S, Blaszkewicz M, Ickstadt K, Hengstler JG, Golka K (2013) Refinement of the prediction of N-acetyltransferase 2 (NAT2) phenotypes with respect to enzyme activity and urinary bladder cancer risk. Arch Toxicol 87(12):2129–2139

    Article  CAS  PubMed  Google Scholar 

  • Selinski S, Blaszkewicz M, Getzmann S, Golka K (2015a) N-Acetyltransferase 2: ultra-slow acetylators enter the stage. Arch Toxicol 89(12):2445–2447

    Article  CAS  PubMed  Google Scholar 

  • Selinski S, Getzmann S, Gajewski PD et al (2015b) The ultra-slow NAT2*6A haplotype is associated with reduced higher cognitive functions in an elderly study group. Arch Toxicol 89(12):2291–2303

    Article  CAS  PubMed  Google Scholar 

  • Shin A, Shrubsole MJ, Rice JM et al (2008) Meat intake, heterocyclic amine exposure, and metabolizing enzyme polymorphisms in relation to colorectal polyp risk. Cancer Epidemiol Biomark Prev 17(2):320–329

    Article  CAS  Google Scholar 

  • van der Hel OL, Peeters PHM, Hein DW et al (2003) NAT2 slow acetylation and GSTM1 null genotypes may increase postmenopausal breast cancer risk in long-term smoking women. Pharmacogenetics 13(7):399–407

    Article  PubMed  Google Scholar 

  • Vatsis KP, Weber WW, Bell DA et al (1995) Nomenclature for N-acetyltransferases. Pharmacogenetics 5(1):1–17

    Article  CAS  PubMed  Google Scholar 

  • Walker K, Ginsberg G, Hattis D, Johns DO, Guyton KZ, Sonawane B (2009) Genetic polymorphism in N-Acetyltransferase (NAT): population distribution of NAT1 and NAT2 activity. J Toxicol Environ Health Part B 12(5–6):440–472

    Article  CAS  Google Scholar 

  • Walraven JM, Zang Y, Trent JO, Hein DW (2008) Structure/function evaluations of single nucleotide polymorphisms in human N-acetyltransferase 2. Curr Drug Metab 9(6):471–486

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang T, Darwin KH, Li H (2010) Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation. Nat Struct Mol Biol 17(11):1352–1357

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Weber WW, Hein DW (1985) N-acetylation pharmacogenetics. Pharmacol Rev 37(1):25–79

    CAS  PubMed  Google Scholar 

  • Zang Y, Doll MA, Zhao S, States JC, Hein DW (2007) Functional characterization of single-nucleotide polymorphisms and haplotypes of human N-acetyltransferase 2. Carcinogenesis 28(8):1665–1671

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang YW, Eom SY, Kim YD et al (2009) Effects of dietary factors and the NAT2 acetylator status on gastric cancer in Koreans. Int J Cancer 125(1):139–145

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmacology and Toxicology and James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY, 40202, USA

    David W. Hein & Mark A. Doll

  2. Department of Pharmacology and Toxicology, University of Louisville Health Sciences Center, Kosair Charities CTR-Room 303, 505 South Hancock Street, Louisville, KY, 40202, USA

    David W. Hein

Authors
  1. David W. Hein
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mark A. Doll
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David W. Hein.

Ethics declarations

Conflict of interest

The authors declares that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hein, D.W., Doll, M.A. Catalytic properties and heat stabilities of novel recombinant human N-acetyltransferase 2 allozymes support existence of genetic heterogeneity within the slow acetylator phenotype. Arch Toxicol 91, 2827–2835 (2017). https://doi.org/10.1007/s00204-017-1989-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00204-017-1989-7

Keywords

Search

Navigation