Impact of N-Acetylation on DNA Damage and Oxidative Stress Responses in Mammalian Cells and Human Hepatocytes Treated with Hydralazine
Abstract
1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. Source and Culture of Cryoplatable Human Hepatocytes
2.3. Construction and Characterization of UV5/Chinese Hamster Ovary (CHO) Cell Lines
2.4. N-Acetyltransferase Assays
2.5. Cell Viability
2.6. DNA Damage Response
2.7. DNA Extraction and Apurinic/Apyrimidinic (AP) Sites Measurement
2.8. Intracellular ROS Detection with DCFDA
2.9. Statistical Analysis
3. Results
3.1. Hydralazine Increases DNA Damage Response in Cryopreserved Human Hepatocytes
3.2. CHO Cells with Rapid NAT2 Acetylator Phenotype Show Higher Hydralazine N-Acetylation Rates Compared with Counterparts with Slow Acetylator Phenotype
3.3. Hydralazine Causes a Concentration-Dependent Increase in γH2AX Expression in CHO Cell Lines
3.4. ROS Levels in CHO Cell Lines Treated with Hydralazine
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
- Sim, E.; Fakis, G.; Laurieri, N.; Boukouvala, S. Arylamine N-acetyltransferases—From drug metabolism and pharmacogenetics to identification of novel targets for pharmacological intervention. Adv. Pharmacol. 2012, 63, 169–205. [Google Scholar] [CrossRef]
- Papanikolaou, G.; Poloni, E.S.; Agúndez, J.A.G.; Teixeira, R.L.F.; Boone, E.C.; Santos, A.R.; Whirl-Carrillo, M.; Sangkuhl, K.; Klein, T.E.; Habil, M.R.; et al. PharmVar GeneFocus: NAT2-Genetic Variation and Updated Nomenclature. Clin. Pharmacol. Ther. 2026, 119, 859–871. [Google Scholar] [CrossRef]
- Sabbagh, A.; Marin, J.; Veyssière, C.; Lecompte, E.; Boukouvala, S.; Poloni, E.S.; Darlu, P.; Crouau-Roy, B. Rapid birth-and-death evolution of the xenobiotic metabolizing NAT gene family in vertebrates with evidence of adaptive selection. BMC Evol. Biol. 2013, 13, 62. [Google Scholar] [CrossRef]
- Han, L.W.; Ryu, R.J.; Cusumano, M.; Easterling, T.R.; Phillips, B.R.; Risler, L.J.; Shen, D.D.; Hebert, M.F. Effect of N-Acetyltransferase 2 Genotype on the Pharmacokinetics of Hydralazine During Pregnancy. J. Clin. Pharmacol. 2019, 59, 1678–1689. [Google Scholar] [CrossRef]
- Sim, E.; Abuhammad, A.; Ryan, A. Arylamine N-acetyltransferases: From drug metabolism and pharmacogenetics to drug discovery. Br. J. Pharmacol. 2014, 171, 2705–2725. [Google Scholar] [CrossRef]
- Browne, J.L.; Klipstein-Grobusch, K.; Franx, A.; Grobbee, D.E. Prevention of Hypertensive Disorders of Pregnancy: A Novel Application of the Polypill Concept. Curr. Cardiol. Rep. 2016, 18, 59. [Google Scholar] [CrossRef][Green Version]
- Moreira-Silva, F.; Camilo, V.; Gaspar, V.; Mano, J.F.; Henrique, R.; Jeronimo, C. Repurposing Old Drugs into New Epigenetic Inhibitors: Promising Candidates for Cancer Treatment? Pharmaceutics 2020, 12, 410. [Google Scholar] [CrossRef]
- Lafi, Z.; Alshaer, W.; Gharaibeh, L.; Alqudah, D.A.; AlQuaissi, B.; Bashaireh, B.; Ibrahim, A.A. Synergistic combination of doxorubicin with hydralazine, and disulfiram against MCF-7 breast cancer cell line. PLoS ONE 2023, 18, e0291981. [Google Scholar] [CrossRef] [PubMed]
- Ruiz-Magana, M.J.; Martinez-Aguilar, R.; Lucendo, E.; Campillo-Davo, D.; Schulze-Osthoff, K.; Ruiz-Ruiz, C. The antihypertensive drug hydralazine activates the intrinsic pathway of apoptosis and causes DNA damage in leukemic T cells. Oncotarget 2016, 7, 21875–21886. [Google Scholar] [CrossRef] [PubMed]
- Castillo-Aguilera, O.; Depreux, P.; Halby, L.; Arimondo, P.B.; Goossens, L. DNA Methylation Targeting: The DNMT/HMT Crosstalk Challenge. Biomolecules 2017, 7, 3. [Google Scholar] [CrossRef] [PubMed]
- Coronel, J.; Cetina, L.; Pacheco, I.; Trejo-Becerril, C.; González-Fierro, A.; de la Cruz-Hernandez, E.; Perez-Cardenas, E.; Taja-Chayeb, L.; Arias-Bofill, D.; Candelaria, M.; et al. A double-blind, placebo-controlled, randomized phase III trial of chemotherapy plus epigenetic therapy with hydralazine valproate for advanced cervical cancer. Preliminary results. Med. Oncol. 2011, 28, S540–S546. [Google Scholar] [CrossRef]
- Liu, Y.C.; Su, C.W.; Lee, R.C.; Liu, C.J.; Huang, Y.H.; Gau, J.P.; Liu, J.H. A clinical trial with valproic acid and hydralazine in combination with gemcitabine and cisplatin followed by doxorubicin and dacarbazine for advanced hepatocellular carcinoma. Asia Pac. J. Clin. Oncol. 2022, 18, 19–27. [Google Scholar] [CrossRef]
- Yang, B.H.; Lin, W.Z.; Chiang, Y.T.; Chen, Y.C.; Chung, C.H.; Chien, W.C.; Shiau, C.Y. Epigenetics-Associated Risk Reduction of Hematologic Neoplasms in a Nationwide Cohort Study: The Chemopreventive and Therapeutic Efficacy of Hydralazine. Front. Oncol. 2022, 12, 809014. [Google Scholar] [CrossRef]
- Garces-Eisele, S.J.; Cedillo-Carvallo, B.; Reyes-Nunez, V.; Estrada-Marín, L.; Vázquez-Pérez, R.; Juárez-Calderón, M.; Guzmán-García, M.O.; Dueñas-González, A.; Ruiz-Argüelles, A. Genetic selection of volunteers and concomitant dose adjustment leads to comparable hydralazine/valproate exposure. J. Clin. Pharm. Ther. 2014, 39, 368–375. [Google Scholar] [CrossRef]
- Allen, C.E.; Doll, M.A.; Hein, D.W. N-Acetyltransferase 2 Genotype-Dependent N-Acetylation of Hydralazine in Human Hepatocytes. Drug Metab. Dispos. 2017, 45, 1276–1281. [Google Scholar] [CrossRef] [PubMed]
- Spinasse, L.B.; Santos, A.R.; Suffys, P.N.; Muxfeldt, E.S.; Salles, G.F. Different phenotypes of the NAT2 gene influences hydralazine antihypertensive response in patients with resistant hypertension. Pharmacogenomics 2014, 15, 169–178. [Google Scholar] [CrossRef] [PubMed]
- Eadon, M.T.; Hein, D.W.; Andersen, M.A.; Chapman, M.B.; Cooper-DeHoff , R.M.; Desta, Z.; Duarte, J.D.; Elchynski, A.L.; Gaedigk, A.; Karol, S.E.; et al. Clinical pharmacogenetics implementation consortium guidelines for NAT2 genotype and hydralazine therapy. Clin. Pharmacol. Ther. 2025, 118, 1430–1436. [Google Scholar] [CrossRef] [PubMed]
- Blanco, M.; Martinez, A.; Urios, A.; Herrera, G.; O’Connor, J.E. Detection of oxidative mutagenesis by isoniazid and other hydrazine derivatives in Escherichia coli WP2 tester strain IC203, deficient in OxyR: Strong protective effects of rat liver S9. Mutat. Res. 1998, 417, 39–46. [Google Scholar] [CrossRef]
- Brambilla, G.; Martelli, A. Genotoxicity and carcinogenicity studies of antihypertensive agents. Mutat. Res. 2006, 612, 115–149. [Google Scholar] [CrossRef] [PubMed]
- Martelli, A.; Allavena, A.; Campart, G.B.; Canonero, R.; Ghia, M.; Mattioli, F.; Mereto, E.; Robbiano, L.; Brambilla, G. In vitro and in vivo testing of hydralazine genotoxicity. J. Pharmacol. Exp. Ther. 1995, 273, 113–120. [Google Scholar] [CrossRef]
- McQueen, C.A.; Maslansky, C.J.; Glowinski, I.B.; Crescenzi, S.B.; Weber, W.W.; Williams, G.M. Relationship between the genetically determined acetylator phenotype and DNA damage induced by hydralazine and 2-aminofluorene in cultured rabbit hepatocytes. Proc. Natl. Acad. Sci. USA 1982, 79, 1269–1272. [Google Scholar] [CrossRef]
- Sedigh-Ardekani, M.; Saadat, M. Evaluation of chromosomal aberrations induced by hydralazine in Chinese hamster ovary cells. Egyp. J. Med. Hum. Genet. 2014, 15, 343–346. [Google Scholar] [CrossRef][Green Version]
- Melton, D.; Lewis, C.D.; Price, N.E.; Gates, K.S. Covalent Adduct Formation between the Antihypertensive Drug Hydralazine and Abasic Sites in Double- and Single-Stranded DNA. Chem. Res. Toxicol. 2014, 27, 2113–2118. [Google Scholar] [CrossRef]
- Habil, M.R.; Salazar-Gonzalez, R.A.; Doll, M.A.; Hein, D.W. 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. 2022, 96, 2999–3012. [Google Scholar] [CrossRef] [PubMed]
- Thompson, L.H.; Rubin, J.S.; Cleaver, J.E.; Whitmore, G.F.; Brookman, K. A screening method for isolating DNA repair-deficient mutants of CHO cells. Somat. Cell Genet. 1980, 6, 391–405. [Google Scholar] [CrossRef] [PubMed]
- Metry, K.J.; Zhao, S.; Neale, J.R.; Doll, M.A.; States, J.C.; McGregor, W.G.; Pierce, W.M., Jr.; Hein, D.W. 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. 2007, 46, 553–563. [Google Scholar] [CrossRef] [PubMed]
- Habil, M.R.; Salazar-González, R.A.; Doll, M.A.; Hein, D.W. Bioactivation, Mutagenicity, DNA Damage, and Oxidative Stress Induced by 3,4-Dimethylaniline. Biomolecules 2024, 14, 1562. [Google Scholar] [CrossRef] [PubMed]
- Lee, S.C.; Jee, S.C.; Kim, M.; Shin, M.K.; Kim, Y.; Sung, J.S. Curcumin Suppresses the Lipid Accumulation and Oxidative Stress Induced by Benzo[a]pyrene Toxicity in HepG2 Cells. Antioxidants 2021, 10, 1314. [Google Scholar] [CrossRef]
- Lemke, L.E.; McQueen, C.A. Acetylation and its role in the mutagenicity of the antihypertensive agent hydralazine. Drug Metab. Dispos. 1995, 23, 559–565. [Google Scholar] [CrossRef]
- Wang, Y.; Liu, L.; Wu, C.; Bulgar, A.; Somoza, E.; Zhu, W.; Gerson, S.L. Direct detection and quantification of abasic sites for in vivo studies of DNA damage and repair. Nucl. Med. Biol. 2009, 36, 975–983. [Google Scholar] [CrossRef]
- Mandi, C.S.; Mahata, T.; Patra, D.; Chakraborty, J.; Bora, A.; Pal, R.; Dutta, S. Cleavage of Abasic Sites in DNA by an Aminoquinoxaline Compound: Augmented Cytotoxicity and DNA Damage in Combination with an Anticancer Drug Chlorambucil in Human Colorectal Carcinoma Cells. ACS Omega 2022, 7, 6488–6501. [Google Scholar] [CrossRef] [PubMed]
- Islam, T.; Nunna, V.; Liyanarachchi, D.P.; Melton, D.; Lewis, C.D.; Gates, K.S. Repurposing the Antihypertensive Agent Hydralazine As an Inhibitor of the Base Excision Repair Enzyme APE1. Chem. Res. Toxicol. 2025, 38, 42–45. [Google Scholar] [CrossRef] [PubMed]
- Boulebd, H.; Khodja, I.A.; Bay, M.V.; Hoa, N.T.; Mechler, A.; Vo, Q.V. Thermodynamic and Kinetic Studies of the Radical Scavenging Behavior of Hydralazine and Dihydralazine: Theoretical Insights. J. Phys. Chem. B 2020, 124, 4123–4131. [Google Scholar] [CrossRef]
- Chang, T.T.; Chen, J.W. Potential Impacts of Hydralazine as a Novel Antioxidant on Cardiovascular and Renal Disease-Beyond Vasodilation and Blood Pressure Lowering. Antioxidants 2022, 11, 2224. [Google Scholar] [CrossRef]
- Chhunchha, B.; Kubo, E.; Krueger, R.R.; Singh, D.P. Hydralazine Revives Cellular and Ocular Lens Health-Span by Ameliorating the Aging and Oxidative-Dependent Loss of the Nrf2-Activated Cellular Stress Response. Antioxidants 2023, 12, 140. [Google Scholar] [CrossRef]
- Taşkıran, A.Ş.; Ergül, M. The Protective Effect of Hydralazine against Hydrogen Peroxide (H2O2)-Induced Oxidative Damage in C6 Glial Cell Line. Turk. J. Sci. Health 2021, 2, 8–15. [Google Scholar]






Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2026 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license.
Share and Cite
Habil, M.R.; Stephens, M.A.; Cass, A.A.; Mittlestat, E.M.; Kwon, D.; Ellison, A.; Kouokam, J.C.; Hein, D.W. Impact of N-Acetylation on DNA Damage and Oxidative Stress Responses in Mammalian Cells and Human Hepatocytes Treated with Hydralazine. Biomolecules 2026, 16, 562. https://doi.org/10.3390/biom16040562
Habil MR, Stephens MA, Cass AA, Mittlestat EM, Kwon D, Ellison A, Kouokam JC, Hein DW. Impact of N-Acetylation on DNA Damage and Oxidative Stress Responses in Mammalian Cells and Human Hepatocytes Treated with Hydralazine. Biomolecules. 2026; 16(4):562. https://doi.org/10.3390/biom16040562
Chicago/Turabian StyleHabil, Mariam R., Makayla A. Stephens, Alexandra A. Cass, Elise M. Mittlestat, Darbie Kwon, Alexandra Ellison, J. Calvin Kouokam, and David W. Hein. 2026. "Impact of N-Acetylation on DNA Damage and Oxidative Stress Responses in Mammalian Cells and Human Hepatocytes Treated with Hydralazine" Biomolecules 16, no. 4: 562. https://doi.org/10.3390/biom16040562
APA StyleHabil, M. R., Stephens, M. A., Cass, A. A., Mittlestat, E. M., Kwon, D., Ellison, A., Kouokam, J. C., & Hein, D. W. (2026). Impact of N-Acetylation on DNA Damage and Oxidative Stress Responses in Mammalian Cells and Human Hepatocytes Treated with Hydralazine. Biomolecules, 16(4), 562. https://doi.org/10.3390/biom16040562

