Abstract
β-naphthylamine (BNA) is an important aromatic amine carcinogen. Current exposures derive primarily from cigarette smoking including e-cigarettes. Occupational and environmental exposure to BNA is associated with urinary bladder cancer which is the fourth most frequent cancer in the United States. N-acetyltransferase 2 (NAT2) is an important metabolizing enzyme for aromatic amines. Previous studies investigated mutagenicity and genotoxicity of BNA in bacteria and in rabbit or rat hepatocytes. However, the effects of human NAT2 genetic polymorphism on N-acetylation and genotoxicity induced by BNA still need to be clarified. We used nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells that were stably transfected with human CYP1A2 and NAT2 alleles: NAT2*4 (reference allele), NAT2*5B (variant slow acetylator allele common in Europe) or NAT2*7B (variant slow acetylator allele common in Asia). BNA N-acetylation was measured both in vitro and in situ via high-performance liquid chromatography (HPLC). Hypoxanthine phosphoribosyl transferase (HPRT) mutations, double-strand DNA breaks, and reactive oxygen species (ROS) were measured as indices of toxicity. NAT2*4 cells showed significantly higher BNA N-acetylation rates followed by NAT2*7B and NAT2*5B. BNA caused concentration-dependent increases in DNA damage and ROS levels. NAT2*7B showed significantly higher levels of HPRT mutants, DNA damage and ROS than NAT2*5B (p < 0.001, p < 0.0001, p < 0.0001 respectively) although both are slow alleles. Our findings suggest that BNA N-acetylation and toxicity are modified by NAT2 polymorphism. Furthermore, they confirm heterogeneity among slow acetylator alleles for BNA metabolism and toxicity supporting differential risk for individuals carrying NAT2*7B allele.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Avirmed S, Khuanbai Y, Sanjaajamts A, Selenge B, Dagvadorj BU, Ohashi M (2021) Modifying effect of smoking on GSTM1 and NAT2 in relation to the risk of bladder cancer in mongolian population: a case-control study. Asian Pac J Cancer Prev 22(8):2479–2485. https://doi.org/10.31557/APJCP.2021.22.8.2479
Baldauf KJ, Salazar-Gonzalez RA, Doll MA, Pierce WM Jr, States JC, Hein DW (2020) Role of human N-acetyltransferase 2 genetic polymorphism on aromatic amine carcinogen-induced DNA damage and mutagenicity in a Chinese hamster ovary cell mutation assay. Environ Mol Mutagen 61(2):235–245. https://doi.org/10.1002/em.22331
Bellamri M, Walmsley SJ, Brown C et al (2022) DNA damage and oxidative stress of tobacco smoke condensate in human bladder epithelial cells. Chem Res Toxicol. https://doi.org/10.1021/acs.chemrestox.2c00153
Bendaly J, Doll MA, Millner LM et al (2009) Differences between human slow N-acetyltransferase 2 alleles in levels of 4-aminobiphenyl-induced DNA adducts and mutations. Mutat Res 671(1–2):13–19
Bjurlin MA, Matulewicz RS, Roberts TR et al (2021) Carcinogen biomarkers in the urine of electronic cigarette users and implications for the development of bladder cancer: a systematic review. Eur Urol Oncol 4(5):766–783. https://doi.org/10.1016/j.euo.2020.02.004
Blackadar CB (2016) Historical review of the causes of cancer. World J Clin Oncol 7(1):54–86. https://doi.org/10.5306/wjco.v7.i1.54
Bonnier F, Keating ME, Wrobel TP et al (2015) Cell viability assessment using the Alamar blue assay: a comparison of 2D and 3D cell culture models. Toxicol in Vitro 29(1):124–131. https://doi.org/10.1016/j.tiv.2014.09.014
Cascorbi I, Brockmoller J, Mrozikiewicz PM, Bauer S, Loddenkemper R, Roots I (1996) Homozygous rapid arylamine N-acetyltransferase (NAT2) genotype as a susceptibility factor for lung cancer. Cancer Res 56(17):3961–3966
Cho HK, Park CG, Shin HJ, Park K, Lim HB (2018) In vitro toxicological activity of particulate matter generated by coal combustion. Environ Toxicol Pharmacol 64:187–195. https://doi.org/10.1016/j.etap.2018.11.003
Czubacka E, Czerczak S (2020) 2-naphthylamine toxicity. Med Pr 71(2):205–220. https://doi.org/10.13075/mp.5893.00921
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
Doll MA, Hein DW (2017) Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes. Arch Toxicol 91(7):2655–2661. https://doi.org/10.1007/s00204-017-1988-8
Dost A, Straughan JK, Sorahan T (2009) Cancer incidence and exposure to 4,4’-methylene-bis-ortho-chloroaniline (MbOCA). Occup Med (oxford, England) 59(6):402–405. https://doi.org/10.1093/occmed/kqp093
El Kawak M, Dhaini HR, Jabbour ME, Moussa MA, El Asmar K, Aoun M (2020) Slow N-acetylation as a possible contributor to bladder carcinogenesis. Mol Carcinog 59(9):1017–1027. https://doi.org/10.1002/mc.23232
Fluckiger-Isler S, Baumeister M, Braun K et al (2004) Assessment of the performance of the Ames II assay: a collaborative study with 19 coded compounds. Mutat Res 558(1–2):181–197. https://doi.org/10.1016/j.mrgentox.2003.12.001
Fontana L, Delort L, Joumard L et al (2009) Genetic polymorphisms in CYP1A1, CYP1B1, COMT, GSTP1 and NAT2 genes and association with bladder cancer risk in a French cohort. Anticancer Res 29(5):1631–1635
Fretland AJ, Leff, MA, Doll MA, Hein DW (2001) Functional characterization of human N-acetyltransferase 2 (NAT2) single nucleotide polymorphisms. Pharmacogenet 11(3):207–215
Fuller TW, Acharya AP, Meyyappan T et al (2018) Comparison of bladder carcinogens in the urine of e-cigarette users versus non e-cigarette using controls. Sci Rep 8(1):507. https://doi.org/10.1038/s41598-017-19030-1
Gatehouse D (2012) Bacterial mutagenicity assays: test methods. Methods Mol Biol 817:21–34. https://doi.org/10.1007/978-1-61779-421-6_2
Gupta RS, Singh B (1982) Mutagenic responses of five independent genetic loci in CHO cells to a variety of mutagens. Development and characteristics of a mutagen screening system based on selection for multiple drug-resistant markers. Mutat Res 94(2):449–466. https://doi.org/10.1016/0027-5107(82)90307-4
Hein DW (2006) N-acetyltransferase 2 genetic polymorphism: effects of carcinogen and haplotype on urinary bladder cancer risk. Oncogene 25:1649–1658. https://doi.org/10.1038/sj.onc.1209374
Hein DW, Doll MA, Fretland AJ et al (2000) Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms. Cancer Epidemiol Biomarkers Prev 9(1):29–42
Hou SM, Falt S, Yang K et al (2001) Differential interactions between GSTM1 and NAT2 genotypes on aromatic DNA adduct level and HPRT mutant frequency in lung cancer patients and population controls. Cancer Epidemiol Biomark Prev 10(2):133–140
Huang Y, Huang S, Wu Y et al (2019) Lipoxygenase protein expression and its effect on oxidative stress caused by benzidine in normal human urothelial cell lines. Int J Toxicol 38(2):121–128. https://doi.org/10.1177/1091581819827495
Humans IWGotEoCRt, (2004) Tobacco smoke and involuntary smoking. IARC Monogr Eval Carcinog Risks Hum 83:1–1438
Inami K, Okazawa M, Mochizuki M (2009) Mutagenicity of aromatic amines and amides with chemical models for cytochrome P450 in Ames assay. Toxicol in Vitro 23(6):986–991. https://doi.org/10.1016/j.tiv.2009.06.025
Kamel AM, Ebid GT, Moussa HS (2015) N-Acetyltransferase 2 (NAT2) polymorphism as a risk modifier of susceptibility to pediatric acute lymphoblastic leukemia. Tumour Biol 36(8):6341–6348. https://doi.org/10.1007/s13277-015-3320-7
Kobets T, Duan JD, Brunnemann KD, Vock E, Deschl U, Williams GM (2019) DNA-damaging activities of twenty-four structurally diverse unsubstituted and substituted cyclic compounds in embryo-fetal chicken livers. Mutat Res Genet Toxicol Environ Mutagen 844:10–24. https://doi.org/10.1016/j.mrgentox.2019.06.004
Krech E, Selinski S, Blaszkewicz M et al (2017) Urinary bladder cancer risk factors in an area of former coal, iron, and steel industries in Germany. J Toxicol Environ Health A 80(7–8):430–438. https://doi.org/10.1080/10937404.2017.1304719
Lee SC, Jee SC, Kim M et al (2021) Curcumin suppresses the lipid accumulation and oxidative stress induced by benzo[a]pyrene toxicity in HepG2 cells. Antioxidants (Basel) 10(8):1314. https://doi.org/10.3390/antiox10081314
Leggett CS, Doll MA, Salazar-Gonzalez 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. https://doi.org/10.1007/s00204-021-03194-x
Liu HE, Hsiao PY, Lee CC, Lee JA, Chen HY (2008) NAT2*7 allele is a potential risk factor for adult brain tumors in Taiwanese population. Cancer Epidemiol Biomarkers Prev 17(3):661–665. https://doi.org/10.1158/1055-9965.EPI-07-2647
Ma QW, Lin GF, Chen JG et al (2004) Polymorphism of N-acetyltransferase 2 (NAT2) gene polymorphism in Shanghai population: occupational and non-occupational bladder cancer patient groups. Biomed Environ Sci 17(3):291–298
Makena P, Chung KT (2007) Evidence that 4-aminobiphenyl, benzidine, and benzidine congeners produce genotoxicity through reactive oxygen species. Environ Mol Mutagen 48(5):404–413. https://doi.org/10.1002/em.20288
McQueen CA, Maslansky CJ, Glowinski IB, Crescenzi SB, Weber WW, Williams GM (1982) Relationship between the genetically determined acetylator phenotype and DNA damage induced by hydralazine and 2-aminofluorene in cultured rabbit hepatocytes. Proc Natl Acad Sci U S A 79(4):1269–1272
McQueen CA, Maslansky CJ, Williams GM (1983) Role of the acetylation polymorphism in determining susceptibility of cultured rabbit hepatocytes to DNA damage by aromatic amines. Cancer Res 43(7):3120–3123
Metry KJ, Zhao S, Neale JR et al (2007) 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine-induced DNA adducts and genotoxicity in chinese hamster ovary (CHO) cells expressing human CYP1A2 and rapid or slow acetylator N-acetyltransferase 2. Mol Carcinog 46(7):553–563. https://doi.org/10.1002/mc.20302[doi]
Metry KJ, Neale JR, Doll MA et al (2010) Effect of rapid human N-acetyltransferase 2 haplotype on DNA damage and mutagenesis induced by 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx). Mutat Res 684(1–2):66–73. https://doi.org/10.1016/j.mrfmmm.2009.12.001
Milovanovic S, Stojanovic J, Pastorino R, Iavicoli I, Boccia S (2017) Occupational exposures and genetic susceptibility to lung cancer and pleural mesothelioma: A systematic review. Epidemiol Biostat Public Health 14:e12559–e12561. https://doi.org/10.2427/12559
Mohammadi H, Roochi MM, Sadeghi M et al (2021) Association of N-acetyltransferases 1 and 2 polymorphisms with susceptibility to head and neck cancers-a meta-analysis, meta-regression, and trial sequential analysis. Medicina (kaunas) 57(10):1095. https://doi.org/10.3390/medicina57101095
Murata M, Tamura A, Tada M, Kawanishi S (2001) Mechanism of oxidative DNA damage induced by carcinogenic 4-aminobiphenyl. Free Radic Biol Med 30(7):765–773. https://doi.org/10.1016/s0891-5849(01)00463-4
Nabavizadeh B, Amend GM, Breyer BN (2021) Workers died of dyes: the discovery of occupational bladder cancers. Urology 154:4–7. https://doi.org/10.1016/j.urology.2021.05.010
Oglesby LA, Hix C, Snow L, MacNair P, Seig M, Langenbach R (1983) Bovine bladder urothelial cell activation of carcinogens to metabolites mutagenic to Chinese hamster V79 cells and Salmonella typhimurium. Cancer Res 43(11):5194–5199
Ohnishi S, Murata M, Kawanishi S (2002) Oxidative DNA damage induced by a metabolite of 2-naphthylamine, a smoking-related bladder carcinogen. Jpn J Cancer Res 93(7):736–743. https://doi.org/10.1111/j.1349-7006.2002.tb01314.x
Orzechowski A, Schrenk D, Bock KW (1992) Metabolism of 1- and 2-naphthylamine in isolated rat hepatocytes. Carcinogenesis 13(12):2227–2232. https://doi.org/10.1093/carcin/13.12.2227
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. https://doi.org/10.1371/journal.pone.0044629
Sabbagh A, Darlu P, Crouau-Roy B, Poloni ES (2011) Arylamine N-acetyltransferase 2 (NAT2) genetic diversity and traditional subsistence: a worldwide population survey. PLoS ONE 6(4):e18507. https://doi.org/10.1371/journal.pone.0018507
Saginala K, Barsouk A, Aluru JS, Rawla P, Padala SA, Barsouk A (2020) Epidemiology of bladder cancer. Med Sci (basel) 8(1):15. https://doi.org/10.3390/medsci8010015
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):7566. https://doi.org/10.1038/s41598-020-64508-0
Selinski S, Blaszkewicz M, Ickstadt K, Hengstler JG, Golka K (2013) Refinement of the prediction of Nacetyltransferase 2 (NAT2) phenotypes with respect to enzyme activity and urinary bladder cancer risk. Arch Toxicol 87(12):2129–2139. https://doi.org/10.1007/s00204-013-1157-7
Talaska G (2003) Aromatic amines and human urinary bladder cancer: exposure sources and epidemiology. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 21(1):29–43. https://doi.org/10.1081/GNC-120021372
Tomioka K, Saeki K, Obayashi K, Kurumatani N (2016) Risk of lung cancer in workers exposed to benzidine and/or beta-naphthylamine: a systematic review and meta-analysis. J Epidemiol 26(9):447–458. https://doi.org/10.2188/jea.JE20150233
Vineis P, Marinelli D, Autrup H et al (2001) Current smoking, occupation, N-acetyltransferase-2 and bladder cancer: a pooled analysis of genotype-based studies. Cancer Epidemiol Biomarkers Prev 10(12):1249–1252
Walia HK, Singh N, Sharma S (2022) Association of NAT-2 gene polymorphisms toward lung cancer susceptibility and prognosis in North Indian patients treated with platinum-based chemotherapy. Pharmacogenomics 23(2):97–118. https://doi.org/10.2217/pgs-2021-0080
Wang S, Hanna D, Sugamori KS, Grant DM (2019) Primary aromatic amines and cancer: novel mechanistic insights using 4-aminobiphenyl as a model carcinogen. Pharmacol Ther 200:179–189. https://doi.org/10.1016/j.pharmthera.2019.05.004
Xu WJ, Wen LP, Jiang XX et al (2017) Association between N-Acetyltransferase 2 polymorphism and bladder cancer risk: a meta-analysis in a single ethnic group. Clin Lab 63(2):287–293. https://doi.org/10.7754/Clin.Lab.2016.160803
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. https://doi.org/10.1093/carcin/bgm085[doi]
Zhou X, Ma Z, Dong D, Wu B (2013) Arylamine N-acetyltransferases: a structural perspective. Br J Pharmacol 169(4):748–760. https://doi.org/10.1111/bph.12182
Zhu K, Xu A, Xia W et al (2021) Association between NAT2 polymorphism and lung cancer risk: a systematic review and meta-analysis. Front Oncol 11:567762. https://doi.org/10.3389/fonc.2021.567762
Acknowledgements
This work was partially supported by United States Public Health Service Grants P30-ES030283 and P42-ES023716. The study is partial fulfillment by Mariam R. Habil for the PhD in pharmacology & toxicology at the University of Louisville.
Funding
This study was funded by NIH, P30-ES030283, P42-ES023716.
Author information
Authors and Affiliations
Contributions
MRH: writing the original draft, doing experiments, data visualization formal analysis, review and editing. RASG: doing experiments, data visualization, formal analysis investigation, writing, review and editing. MAD: formal analysis, investigation, writing, review and editing. DWH: conceptualization, methodology, validation, formal analysis, resources, writing, review and editing, visualization, supervision.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Habil, M.R., Salazar-González, R.A., Doll, M.A. et al. Differences in β-naphthylamine metabolism and toxicity in Chinese hamster ovary cell lines transfected with human CYP1A2 and NAT2*4, NAT2*5B or NAT2*7B N-acetyltransferase 2 haplotypes. Arch Toxicol 96, 2999–3012 (2022). https://doi.org/10.1007/s00204-022-03367-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00204-022-03367-2