Abstract
Benzidine undergoes N-acetylation and following CYP1A2-catalyzed N-hydroxylation undergoes O-acetylation catalyzed by N-acetyltransferase 1 (NAT1). Benzidine exposure is associated with urinary bladder cancer but the effect of NAT1 genetic polymorphism on individual risk remains unclear. We used Chinese hamster ovary (CHO) cells transfected with human CYP1A2 and NAT1*4 allele (reference) or NAT1*14B (variant) to investigate the effects of dose and NAT1 polymorphism on benzidine metabolism and genotoxicity. Rates of benzidine N-acetylation in vitro were higher in CHO cells transfected with NAT1*4 compared to NAT1*14B. CHO cells transfected with NAT1*14B exhibited greater N-acetylation rates in situ than cells transfected with NAT1*4 at low doses of benzidine expected with environmental exposures but not at higher doses. NAT1*14B exhibited over tenfold lower apparent KM which resulted in higher intrinsic clearance for benzidine N-acetylation compared to CHO cells transfected with NAT1*4. Benzidine-induced hypoxanthine phosphoribosyl transferase (HPRT) mutations were higher in CHO cells transfected with NAT1*14B than with NAT1*4 (p < 0.001). Benzidine caused concentration-dependent increase in γ-H2AX signal (indicative of DNA double-strand breaks) in CHO cells transfected with NAT1*4 or NAT1*14B. CHO cells transfected with NAT1*14B exhibited significantly higher level of DNA damage than with NAT1*4 (p < 0.0001). Benzidine-induced ROS did not differ significantly (p > 0.05) between CHO cells transfected with NAT1*4 or NAT1*14B except at 50 μM. Levels of benzidine-induced DNA damage and reactive oxygen species (ROS) showed strong dose-dependent correlation. Our findings support human studies associating NAT1*14B with increased incidence or severity of urinary bladder cancer in workers exposed to benzidine.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The datasets for this study are available from the corresponding author upon request.
References
Agency for Toxic Substances and Disease Registry (ATSDR) (2001) Toxicological profile for Benzidine. Update. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service. http://www.atsdr.cdc.gov/toxprofiles/tp62.pdf. Accessed 19 Jan 2023
Agnello M, Fontana M (2015) Survey on European studies of the chemical characterisation of tattoo ink products and the measurement of potentially harmful ingredients. Curr Probl Dermatol 48:142–151
Avinash BM, Raman JD, Kaag MG (2021) Bladder Cancer: Overview, Epidemiology, Initial Presentation and Diagnosis. In: Trabulsi EJ, Lallas CD, Lizardi-Calvaresi AE (eds) Chemotherapy and immunotherapy in urologic oncology: a guide for the advanced practice provider. Springer International Publishing, Cham, pp 141–157
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
Ben Khedher S, Neri M, Guida F et al (2018) Occupational exposure to textile dust and lung cancer risk: results from the ICARE study. Am J Ind Med 61(3):216–228. https://doi.org/10.1002/ajim.22799
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
Bouchardy C et al. (1998) N-acetyltransferase NAT1 and NAT2 genotypes and lung cancer risk. Pharmacogenetics 8(4):291–298
Carreón T, LeMasters GK, Ruder AM, Schulte PA (2006) The genetic and environmental factors involved in benzidine metabolism and bladder carcinogenesis in exposed workers. Front Biosci 11:2889–2902
Case RA, Hosker ME, Mc DD, Pearson JT (1954) Tumours of the urinary bladder in workmen engaged in the manufacture and use of certain dyestuff intermediates in the British chemical industry. I. The role of aniline, benzidine, alpha-naphthylamine, and beta-naphthylamine. Br J Ind Med 11(2):75–104. https://doi.org/10.1136/oem.11.2.75
Ching Chen S, Hseu YC, Sung JC, Chen CH, Chen LC, Chung KT (2011) Induction of DNA damage signaling genes in benzidine-treated HepG2 cells. Environ Mol Mutagen 52(8):664–672. https://doi.org/10.1002/em.20669
Chung KT, Chen SC, Claxton LD (2006) Review of the Salmonella typhimurium mutagenicity of benzidine, benzidine analogues, and benzidine-based dyes. Mutat Res 612(1):58–76. https://doi.org/10.1016/j.mrrev.2005.08.001
Ciocan C, Godono A, Franco N et al (2022) Mortality from bladder cancer in dyestuff workers exposed to aromatic amines: a 73-year follow-up. Med Lav 113(2):e2022017. https://doi.org/10.23749/mdl.v113i2.12893
Dapson RW (2009) Benzidine-based dyes: effects of industrial practices, regulations, and world trade on the biological stains market. Biotech Histochem 84(3):95–100. https://doi.org/10.1080/10520290902879730
Degen GH, Schlattjan JH, Mahler S, Follmann W, Golka K (2004) Comparative metabolic activation of benzidine and N-acetylbenzidine by prostaglandin H synthase. Toxicol Lett 151(1):135–142. https://doi.org/10.1016/j.toxlet.2003.11.015
Ding D, Liu Z, Zhao L, Geng H, Liang Z, Yu D (2019) Role of PI3K/Akt pathway in Benzidine-induced proliferation in SV-40 immortalized human uroepithelial cell. Transl Cancer Res 8(4):1301–1310. https://doi.org/10.21037/tcr.2019.07.14
Doll MA, Hein DW (2022) 560G>A (rs4986782) (R187Q) Single nucleotide polymorphism in arylamine N-Acetyltransferase 1 increases affinity for the aromatic amine carcinogens 4-Aminobiphenyl and N-Hydroxy-4-Aminobiphenyl: implications for cancer risk assessment. Front Pharmacol 13:820082. https://doi.org/10.3389/fphar.2022.820082
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
Febriana SA, Jungbauer F, Soebono H, Coenraads PJ (2012) Occupational contact allergy caused by benzidine in three tannery workers. Contact Dermatitis 66(6):345–346. https://doi.org/10.1111/j.1600-0536.2012.02015.x
Fretland AJ, Doll MA, Leff MA, Hein DW (2001) Functional characterization of nucleotide polymorphisms in the coding region of N-acetyltransferase 1. Pharmacogenetics 11(6):511–520. https://doi.org/10.1097/00008571-200108000-00006
Ghanghro L (2021) Chemical risk factors of primary liver cancer: a short comment [Letter]. Hepat Med 13:121–122. https://doi.org/10.2147/HMER.S350076
Guo WC, Lin GF, Chen JG, Golka K, Shen JH (2004) Polymorphism in the N-acetyltransferase 1 alleles NAT1*10 and NAT1*14A and cytological gradings of exfoliated urothelial cells in benzidine-exposed Chinese workers: discussion of ethnic differences. Arch Toxicol 78(8):425–429
Habil MR, Doll MA, Hein DW (2022a) Acetyl coenzyme A kinetic studies on N-acetylation of environmental carcinogens by human N-acetyltransferase 1 and its NAT1*14B variant. Front Pharmacol 13:931323. https://doi.org/10.3389/fphar.2022.931323
Habil MR, Salazar-Gonzalez RA, Doll MA, Hein DW (2022b) Differences in beta-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(11):2999–3012. https://doi.org/10.1007/s00204-022-03367-2
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
Hughes NC, Janezic SA, McQueen KL et al (1998) Identification and characterization of variant alleles of human acetyltransferase NAT1 with defective function using p-aminosalicylate as an in-vivo and in-vitro probe. Pharmacogenetics 8(1):55–66. https://doi.org/10.1097/00008571-199802000-00008
Johnson GE, Quick EL, Parry EM, Parry JM (2010) Metabolic influences for mutation induction curves after exposure to Sudan-1 and para red. Mutagenesis 25(4):327–333. https://doi.org/10.1093/mutage/geq009
Lakshmi VM, Zenser TV, Goldman HD et al (1995) The role of acetylation in benzidine metabolism and DNA adduct formation in dog and rat liver. Chem Res Toxicol 8(5):711–720. https://doi.org/10.1021/tx00047a011
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). https://doi.org/10.3390/antiox10081314
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. https://doi.org/10.1007/s00204-020-02901-4
Letasiova S, Medve’ova A, Sovcikova A et al (2012) Bladder cancer, a review of the environmental risk factors. Environ Health 11(Suppl 1):S11. https://doi.org/10.1186/1476-069X-11-S1-S11
Lim H-H, Shin H-S (2015) Identification and quantification of phthalates, PAHs, amines, phenols, and metals in tattoo. Bull Korean Chem Soc 36(8):2039–2050. https://doi.org/10.1002/bkcs.10395
Lowry LK, Tolos WP, Boeniger MF et al (1980) Chemical monitoring of urine from workers potentially exposed to benzidine-derived azo dyes. Toxicol Lett 7:29–36
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
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. https://doi.org/10.1097/fpc.0000000000000062
Millerick-May ML, Wang L, Rice C, Rosenman KD (2021) Ongoing risk of bladder cancer among former workers at the last benzidine manufacturing facility in the USA. Occup Environ Med 78(9):625. https://doi.org/10.1136/oemed-2020-106431
Millner LM, Doll MA, Cai J, States JC, Hein DW (2012) Phenotype of the most common “slow acetylator” arylamine N-acetyltransferase 1 genetic variant (NAT1*14B) is substrate-dependent. Drug Metab Dispos 40(1):198–204. https://doi.org/10.1124/dmd.111.041855
Miyakawa M, Tachibana M, Miyakawa A et al (2001) Re-evaluation of the latent period of bladder cancer in dyestuff-plant workers in Japan. Int J Urol 8(8):423–430. https://doi.org/10.1046/j.1442-2042.2001.00342.x
Naito S, Tanaka K, Koga H, Kotoh S, Hirohata T, Kumazawa J (1995) Cancer occurrence among dyestuff workers exposed to aromatic amines. A Long Term Follow-up Study. Cancer 76(8):1445–1452. https://doi.org/10.1002/1097-0142(19951015)76:8%3c1445::aid-cncr2820760823%3e3.0.co;2-r
Rothman N, Bhatnagar VK, Hayes RB et al (1996) The impact of interindividual variation in NAT2 activity on benzidine urinary metabolites and urothelial DNA adducts in exposed workers. Proc Natl Acad Sci U S A 93(10):5084–5089. https://doi.org/10.1073/pnas.93.10.5084
Schneider K, Hafner C, Jager I (2004) Mutagenicity of textile dye products. J Appl Toxicol 24(2):83–91. https://doi.org/10.1002/jat.953
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
Yassine IA, Kobeissi L, Jabbour ME, Dhaini HR (2012) N-Acetyltransferase 1 (NAT1) genotype: a risk factor for urinary bladder cancer in a lebanese population. J Oncol 2012:512976
Zenser TV, Lakshmi VM, Rustan TD et al (1996) Human N-acetylation of benzidine: role of NAT1 and NAT2. Cancer Res 56(17):3941–3947
Zhu Y, Hein DW (2008) Functional effects of single nucleotide polymorphisms in the coding region of human N-acetyltransferase 1. Pharmacogenomics J 8(5):339–348. https://doi.org/10.1038/sj.tpj.6500483
Acknowledgements
The results of this study were submitted in partial fulfillment for the PhD in pharmacology and toxicology at the University of Louisville by Mariam Habil. The authors thank laboratory colleagues: Mark A. Doll, Raúl A. Salazar González, and James T.F. Wise for their contributions towards initial construction and authentication of the CHO cells. The work was partially supported by United States Public Health Service grants P42-ES023716, P20-GM113226 and P30-ES030283.
Author information
Authors and Affiliations
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 (e.g. a society or other partner) 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., Hein, D.W. Effects of dose and human N-acetyltransferase 1 genetic polymorphism in benzidine metabolism and genotoxicity. Arch Toxicol 97, 1765–1772 (2023). https://doi.org/10.1007/s00204-023-03497-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00204-023-03497-1