Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes

Abstract

Genetic polymorphisms in human N-acetyltransferase 2 (NAT2) modify the metabolism of numerous drugs and carcinogens. These genetic polymorphisms modify both drug efficacy and toxicity and cancer risk associated with carcinogen exposure. Previous studies have suggested phenotypic heterogeneity among different NAT2 slow acetylator genotypes. NAT2 phenotype was investigated in vitro and in situ in samples of human hepatocytes obtained from various NAT2 slow and intermediate NAT2 acetylator genotypes. NAT2 gene dose response (NAT2*5B/*5B > NAT2*5B/*6A > NAT2*6A/*6A) was observed towards the N-acetylation of the NAT2-specific drug sulfamethazine by human hepatocytes both in vitro and in situ. N-acetylation of 4-aminobiphenyl, an arylamine carcinogen substrate for both N-acetyltransferase 1 and NAT2, showed the same trend both in vitro and in situ although the differences were not significant (p > 0.05). The N-acetylation of the N-acetyltransferase 1-specific substrate p-aminobenzoic acid did not follow this trend. In comparisons of NAT2 intermediate acetylator genotypes, differences in N-acetylation between NAT2*4/*5B and NAT2*4/*6B hepatocytes were not observed in vitro or in situ towards any of these substrates. These results further support phenotypic heterogeneity among NAT2 slow acetylator genotypes, consistent with differential risks of drug failure or toxicity and cancer associated with carcinogen exposure.

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References

  1. 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

    CAS  Article  Google Scholar 

  2. 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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Bolt HM, Selinski S, Dannappel D, Blaszkewicz M, Golka K (2005) Re-investigation of the concordance of human NAT2 phenotypes and genotypes. Arch Toxicol 79(4):196–200

    CAS  Article  PubMed  Google Scholar 

  4. Cascorbi I, Drakoulis N, Brockmoller J, Maurer A, Sperling K, Roots I (1995) Arylamine N-acetyltransferase (NAT2) mutations and their allelic linkage in unrelated Caucasian individuals: correlation with phenotypic activity. Am J Hum Genet 57(3):581–592

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 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 

  6. 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 

  7. Doll MA, Hein DW (2001) Comprehensive human NAT2 genotype method using single nucleotide polymorphism-specific polymerase chain reaction primers and fluorogenic probes. Anal Biochem 288(1):106–108

    CAS  Article  PubMed  Google Scholar 

  8. 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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  11. 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 

  12. 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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 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)

    Google Scholar 

  14. Hein DW, Doll MA (2012) Accuracy of various human NAT2 SNP genotyping panels to infer rapid, intermediate and slow acetylator phenotypes. Pharmacogenomics 13(1):31–41

    CAS  Article  PubMed  Google Scholar 

  15. 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

    CAS  Article  PubMed  Google Scholar 

  16. 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

    CAS  Article  PubMed  Google Scholar 

  17. 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

    CAS  Article  PubMed  Google Scholar 

  18. 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 

  19. 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

    CAS  Article  PubMed  Google Scholar 

  20. 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

    CAS  Article  PubMed  Google Scholar 

  21. 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 

  22. Meisel P, Arndt D, Scheuch E, Klebingat KJ, Siegmund W (2001) Prediction of metabolic activity from genotype: the gene-dose effect of N-acetyltransferase. Ther Drug Monit 23(1):9–14

    CAS  Article  PubMed  Google Scholar 

  23. 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

    CAS  Article  PubMed  Google Scholar 

  24. 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

    CAS  Article  PubMed  Google Scholar 

  25. Rothman N, Hayes RB, Bi W et al (1993) Correlation between N-acetyltransferase activity and NAT2 genotype in Chinese males. Pharmacogenetics 3(5):250–255

    CAS  Article  PubMed  Google Scholar 

  26. 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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 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

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  29. 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

    CAS  Article  PubMed  Google Scholar 

  30. 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

    CAS  Article  Google Scholar 

  31. Smith CA, Wadelius M, Gough AC, Harrison DJ, Wolf CR, Rane A (1997) A simplified assay for the arylamine N-acetyltransferase 2 polymorphism validated by phenotyping with isoniazid. J Med Genet 34(9):758–760

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 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 

  33. 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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  35. Zabost A, Brzezinska S, Kozinska M et al (2013) Correlation of N-acetyltransferase 2 genotype with isoniazid acetylation in Polish tuberculosis patients. Biomed Res Int 2013:853602. doi:10.1155/2013/853602

    Article  PubMed  PubMed Central  Google Scholar 

  36. Zang Y, Zhao S, Doll MA, States JC, Hein DW (2004) The T341C (Ile114Thr) polymorphism of N-acetyltransferase 2 yields slow acetylator phenotype by enhanced protein degradation. Pharmacogenetics 14(11):717–723

    CAS  Article  PubMed  Google Scholar 

  37. 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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Timothy Moeller and Bioreclamation IVT (Baltimore, MD) for their valuable contributions towards this study.

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Correspondence to David W. Hein.

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Doll, M.A., Hein, D.W. Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes. Arch Toxicol 91, 2655–2661 (2017). https://doi.org/10.1007/s00204-017-1988-8

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Keywords

  • N-acetyltransferase 2
  • Slow acetylator genotype
  • Human hepatocytes
  • Sulfamethazine
  • 4-Aminobiphenyl