{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,11]],"date-time":"2026-05-11T23:40:18Z","timestamp":1778542818408,"version":"3.51.4"},"update-to":[{"DOI":"10.1371\/journal.pntd.0013647","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2025,10,30]],"date-time":"2025-10-30T00:00:00Z","timestamp":1761782400000}}],"reference-count":42,"publisher":"Public Library of Science (PLoS)","issue":"10","license":[{"start":{"date-parts":[[2025,10,24]],"date-time":"2025-10-24T00:00:00Z","timestamp":1761264000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100006492","name":"Division of Intramural Research, National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01AI170769"],"award-info":[{"award-number":["R01AI170769"]}],"id":[{"id":"10.13039\/100006492","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100006492","name":"Division of Intramural Research, National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R21AI174051"],"award-info":[{"award-number":["R21AI174051"]}],"id":[{"id":"10.13039\/100006492","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosntds.org"],"crossmark-restriction":false},"short-container-title":["PLoS Negl Trop Dis"],"abstract":"<jats:p>Nitroaromatic drugs are of critical importance for the treatment of trypanosome infections in Africa and the Americas. Fexinidazole recently joined benznidazole and nifurtimox in this family when it was approved as the first oral monotherapy against Human African trypanosomiasis (HAT). Nitroaromatic prodrugs are bioactivated by the trypanosome-specific type I nitroreductase (NTR) that renders the compounds trypanocidal. The trypanocidal activity of nitroaromatic drugs has been proposed to arise from the formation of reactive oxygen species (ROS) and DNA damage. However, the specific cytotoxic effects of nitroaromatic drugs had not been thoroughly interrogated. Here we evaluate and compare the effects of clinically relevant anti-trypanosome nitroaromatic drugs using cell biology phenotypes including cell cycle progression, DNA synthesis, and DNA damage formation in the African trypanosome. We observed that fexinidazole induced cytotoxicity is distinct from related nitroaromatic drugs in its inhibition of DNA synthesis and the timing and magnitude of DNA damage formation. These findings highlight the relationship between nitroaromatic drug treatments, DNA damage formation, and ROS activation. Deconvolving the relationship between anti-parasitic drugs and the molecular basis of their cytotoxic outcomes will support future mechanistic understanding and enable improved drug design.<\/jats:p>","DOI":"10.1371\/journal.pntd.0013647","type":"journal-article","created":{"date-parts":[[2025,10,24]],"date-time":"2025-10-24T17:46:51Z","timestamp":1761328011000},"page":"e0013647","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":2,"title":["Fexinidazole results in specific effects on DNA synthesis and DNA damage in the African trypanosome"],"prefix":"10.1371","volume":"19","author":[{"given":"Kenna E.","family":"Berg","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Indea","family":"Rogers","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hayley M.","family":"Ramirez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Julian","family":"Cornejo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ignacio M.","family":"Durante","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5995-9329","authenticated-orcid":true,"given":"Galadriel","family":"Hovel-Miner","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2025,10,24]]},"reference":[{"issue":"2","key":"pntd.0013647.ref001","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1007\/s40265-019-1051-6","article-title":"Fexinidazole: First Global Approval","volume":"79","author":"ED Deeks","year":"2019","journal-title":"Drugs"},{"issue":"2","key":"pntd.0013647.ref002","article-title":"New WHO guidelines for treating rhodesiense human African trypanosomiasis: expanded indications for fexinidazole and pentamidine","volume":"25","author":"AK Lindner","year":"2024","journal-title":"Lancet Infect Dis"},{"issue":"2","key":"pntd.0013647.ref003","article-title":"New WHO guidelines for treatment of gambiense human African trypanosomiasis including fexinidazole: substantial changes for clinical practice","volume":"20","author":"AK Lindner","year":"2019","journal-title":"Lancet Infect Dis"},{"issue":"1","key":"pntd.0013647.ref004","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1080\/00034983.1983.11811668","article-title":"The activity of fexinidazole (HOE 239) against experimental infections with Trypanosoma cruzi, trichomonads and Entamoeba histolytica","volume":"77","author":"W Raether","year":"1983","journal-title":"Ann Trop Med Parasitol"},{"issue":"1","key":"pntd.0013647.ref005","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/0006-2952(86)90565-4","article-title":"The biological properties of reduced nitroheterocyclics and possible underlying biochemical mechanisms","volume":"35","author":"GF Whitmore","year":"1986","journal-title":"Biochem Pharmacol"},{"issue":"13","key":"pntd.0013647.ref006","doi-asserted-by":"crossref","first-page":"5022","DOI":"10.1073\/pnas.0711014105","article-title":"A mechanism for cross-resistance to nifurtimox and benznidazole in trypanosomes","volume":"105","author":"SR Wilkinson","year":"2008","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"6","key":"pntd.0013647.ref007","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1016\/j.pt.2014.04.003","article-title":"Nitro drugs for the treatment of trypanosomatid diseases: past, present, and future prospects","volume":"30","author":"S Patterson","year":"2014","journal-title":"Trends Parasitol"},{"issue":"15","key":"pntd.0013647.ref008","doi-asserted-by":"crossref","first-page":"13088","DOI":"10.1074\/jbc.M111.230847","article-title":"Nifurtimox activation by trypanosomal type I nitroreductases generates cytotoxic nitrile metabolites","volume":"286","author":"BS Hall","year":"2011","journal-title":"J Biol Chem"},{"issue":"1","key":"pntd.0013647.ref009","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1128\/AAC.05135-11","article-title":"Activation of benznidazole by trypanosomal type I nitroreductases results in glyoxal formation","volume":"56","author":"BS Hall","year":"2012","journal-title":"Antimicrob Agents Chemother"},{"issue":"2","key":"pntd.0013647.ref010","doi-asserted-by":"crossref","first-page":"674","DOI":"10.1111\/mmi.14763","article-title":"Unraveling the antitrypanosomal mechanism of benznidazole and related 2-nitroimidazoles: From prodrug activation to DNA damage","volume":"116","author":"A Dattani","year":"2021","journal-title":"Mol Microbiol"},{"issue":"11","key":"pntd.0013647.ref011","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1007\/s40267-019-00672-2","article-title":"Fexinidazole in human African trypanosomiasis: a profile of its use","volume":"35","author":"ED Deeks","year":"2019","journal-title":"Drugs Ther Perspect"},{"issue":"3","key":"pntd.0013647.ref012","doi-asserted-by":"crossref","first-page":"625","DOI":"10.1093\/jac\/dkv376","article-title":"Nitroheterocyclic drug resistance mechanisms in Trypanosoma brucei","volume":"71","author":"S Wyllie","year":"2016","journal-title":"J Antimicrob Chemother"},{"issue":"7","key":"pntd.0013647.ref013","doi-asserted-by":"crossref","first-page":"2893","DOI":"10.1128\/AAC.00332-10","article-title":"Cross-resistance to nitro drugs and implications for treatment of human African trypanosomiasis","volume":"54","author":"AY Sokolova","year":"2010","journal-title":"Antimicrob Agents Chemother"},{"issue":"11","key":"pntd.0013647.ref014","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pntd.0006980","article-title":"Insights into antitrypanosomal drug mode-of-action from cytology-based profiling","volume":"12","author":"JA Thomas","year":"2018","journal-title":"PLoS Negl Trop Dis"},{"issue":"1","key":"pntd.0013647.ref015","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1016\/S0166-6851(99)00002-X","article-title":"A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei","volume":"99","author":"E Wirtz","year":"1999","journal-title":"Mol Biochem Parasitol"},{"issue":"2","key":"pntd.0013647.ref016","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1016\/0169-4758(94)90402-2","article-title":"Axenic culture of African trypanosome bloodstream forms","volume":"10","author":"H Hirumi","year":"1994","journal-title":"Parasitol Today"},{"key":"pntd.0013647.ref017","first-page":"301","article-title":"Analysis of cell cycle by flow cytometry","volume":"281","author":"P Pozarowski","year":"2004","journal-title":"Methods Mol Biol"},{"issue":"8","key":"pntd.0013647.ref018","article-title":"Telomere length affects the frequency and mechanism of antigenic variation in Trypanosoma brucei","volume":"8","author":"GA Hovel-Miner","year":"2012","journal-title":"PLoS Pathog"},{"issue":"3","key":"pntd.0013647.ref019","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1007\/BF01190834","article-title":"Bromodeoxyuridine immunohistochemical determination of the lengths of the cell cycle and the DNA-synthetic phase for an anatomically defined population","volume":"18","author":"RS Nowakowski","year":"1989","journal-title":"J Neurocytol"},{"key":"pntd.0013647.ref020","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1007\/978-1-0716-2736-5_16","article-title":"BrdU Incorporation Assay to Analyze the Entry into S Phase","volume":"2579","author":"J Yu","year":"2022","journal-title":"Methods Mol Biol"},{"issue":"6","key":"pntd.0013647.ref021","doi-asserted-by":"crossref","first-page":"756","DOI":"10.1111\/jeu.12408","article-title":"Differences in the Detection of BrdU\/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids","volume":"64","author":"MS da Silva","year":"2017","journal-title":"J Eukaryot Microbiol"},{"issue":"10","key":"pntd.0013647.ref022","doi-asserted-by":"crossref","first-page":"1143","DOI":"10.1017\/S0031182021000317","article-title":"Effects of trypanocidal drugs on DNA synthesis: new insights into melarsoprol growth inhibition","volume":"148","author":"S Larson","year":"2021","journal-title":"Parasitology"},{"issue":"1","key":"pntd.0013647.ref023","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1016\/j.molbiopara.2012.01.008","article-title":"Trypanosomal histone \u03b3H2A and the DNA damage response","volume":"183","author":"L Glover","year":"2012","journal-title":"Mol Biochem Parasitol"},{"issue":"1","key":"pntd.0013647.ref024","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.molbiopara.2007.01.017","article-title":"Cell cycle regulation in Trypanosoma brucei","volume":"153","author":"TC Hammarton","year":"2007","journal-title":"Molecular and Biochemical Parasitology"},{"issue":"1","key":"pntd.0013647.ref025","doi-asserted-by":"crossref","DOI":"10.1371\/journal.ppat.1003886","article-title":"Regulators of Trypanosoma brucei cell cycle progression and differentiation identified using a kinome-wide RNAi screen","volume":"10","author":"NG Jones","year":"2014","journal-title":"PLoS Pathog"},{"issue":"10","key":"pntd.0013647.ref026","doi-asserted-by":"crossref","first-page":"6298","DOI":"10.1016\/S0021-9258(20)82140-1","article-title":"Reduction of nifurtimox and nitrofurantoin to free radical metabolites by rat liver mitochondria. Evidence of an outer membrane-located nitroreductase","volume":"259","author":"SN Moreno","year":"1984","journal-title":"Journal of Biological Chemistry"},{"issue":"15","key":"pntd.0013647.ref027","doi-asserted-by":"crossref","first-page":"11596","DOI":"10.1074\/jbc.M109.076984","article-title":"Trypanosoma cruzi induces the reactive oxygen species-PARP-1-RelA pathway for up-regulation of cytokine expression in cardiomyocytes","volume":"285","author":"X Ba","year":"2010","journal-title":"J Biol Chem"},{"issue":"11","key":"pntd.0013647.ref028","doi-asserted-by":"crossref","first-page":"3554","DOI":"10.1021\/ac050306u","article-title":"Probing the kinetics of SYTOX Orange stain binding to double-stranded DNA with implications for DNA analysis","volume":"77","author":"X Yan","year":"2005","journal-title":"Anal Chem"},{"issue":"7","key":"pntd.0013647.ref029","doi-asserted-by":"crossref","first-page":"2893","DOI":"10.1128\/AAC.00332-10","article-title":"Cross-resistance to nitro drugs and implications for treatment of human African trypanosomiasis","volume":"54","author":"AY Sokolova","year":"2010","journal-title":"Antimicrob Agents Chemother"},{"issue":"8","key":"pntd.0013647.ref030","doi-asserted-by":"crossref","first-page":"4362","DOI":"10.1128\/AAC.02754-13","article-title":"Antitrypanosomal activity of fexinidazole metabolites, potential new drug candidates for Chagas disease","volume":"58","author":"MT Bahia","year":"2014","journal-title":"Antimicrob Agents Chemother"},{"issue":"11","key":"pntd.0013647.ref031","doi-asserted-by":"crossref","first-page":"1320","DOI":"10.1017\/S0031182021001001","article-title":"Combination therapy using nitro compounds improves the efficacy of experimental Chagas disease treatment","volume":"148","author":"AL Mazzeti","year":"2021","journal-title":"Parasitology"},{"issue":"23","key":"pntd.0013647.ref032","doi-asserted-by":"crossref","first-page":"4454","DOI":"10.2174\/0929867325666180426164352","article-title":"Current and Future Prospects of Nitro-compounds as Drugs for Trypanosomiasis and Leishmaniasis","volume":"26","author":"S Patterson","year":"2019","journal-title":"Curr Med Chem"},{"issue":"3","key":"pntd.0013647.ref033","doi-asserted-by":"crossref","first-page":"625","DOI":"10.1093\/jac\/dkv376","article-title":"Nitroheterocyclic drug resistance mechanisms in Trypanosoma brucei","volume":"71","author":"S Wyllie","year":"2016","journal-title":"J Antimicrob Chemother"},{"issue":"11","key":"pntd.0013647.ref034","doi-asserted-by":"crossref","first-page":"4540","DOI":"10.1021\/bi00437a004","article-title":"Oxygen transfer from the nitro group of a nitroaromatic radiosensitizer to a DNA sugar damage product","volume":"28","author":"LS Kappen","year":"1989","journal-title":"Biochemistry"},{"key":"pntd.0013647.ref035","doi-asserted-by":"crossref","first-page":"105345","DOI":"10.1016\/j.aquatox.2019.105345","article-title":"Nitroaromatic compounds damage the DNA of zebrafish embryos (Danio rerio)","volume":"217","author":"D Koske","year":"2019","journal-title":"Aquat Toxicol"},{"issue":"1","key":"pntd.0013647.ref036","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1038\/bjc.1979.144","article-title":"Inhibition of DNA synthesis by nitroheterocycles. I. Correlation with half-wave reduction potential","volume":"40","author":"PL Olive","year":"1979","journal-title":"Br J Cancer"},{"issue":"12","key":"pntd.0013647.ref037","first-page":"1244","article-title":"Repair of 8-oxoguanine in Saccharomyces cerevisiae: interplay of DNA repair and replication mechanisms","volume":"32","author":"S Boiteux","year":"2002","journal-title":"Free Radic Biol Med"},{"issue":"8","key":"pntd.0013647.ref038","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0042484","article-title":"Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi","volume":"7","author":"C Furtado","year":"2012","journal-title":"PLoS One"},{"issue":"6","key":"pntd.0013647.ref039","article-title":"Oxidative stress and DNA lesions: the role of 8-oxoguanine lesions in Trypanosoma cruzi cell viability","volume":"7","author":"PHN Aguiar","year":"2013","journal-title":"PLoS Negl Trop Dis"},{"issue":"8","key":"pntd.0013647.ref040","doi-asserted-by":"crossref","first-page":"1167","DOI":"10.1016\/j.freeradbiomed.2008.07.018","article-title":"DNA damage-induced reactive oxygen species (ROS) stress response in Saccharomyces cerevisiae","volume":"45","author":"LA Rowe","year":"2008","journal-title":"Free Radic Biol Med"},{"issue":"1","key":"pntd.0013647.ref041","article-title":"DNA damage induces reactive oxygen species generation through the H2AX-Nox1\/Rac1 pathway","volume":"3","author":"MA Kang","year":"2012","journal-title":"Cell Death Dis"},{"issue":"8","key":"pntd.0013647.ref042","doi-asserted-by":"crossref","first-page":"2607","DOI":"10.1073\/pnas.86.8.2607","article-title":"Trypanothione is the primary target for arsenical drugs against African trypanosomes","volume":"86","author":"AH Fairlamb","year":"1989","journal-title":"Proc Natl Acad Sci U S A"}],"updated-by":[{"DOI":"10.1371\/journal.pntd.0013647","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2025,10,30]],"date-time":"2025-10-30T00:00:00Z","timestamp":1761782400000}}],"container-title":["PLOS Neglected Tropical Diseases"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pntd.0013647","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,30]],"date-time":"2025-10-30T17:53:30Z","timestamp":1761846810000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pntd.0013647"}},"subtitle":[],"editor":[{"given":"Brice","family":"Rotureau,","sequence":"first","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2025,10,24]]},"references-count":42,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2025,10,24]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pntd.0013647","relation":{},"ISSN":["1935-2735"],"issn-type":[{"value":"1935-2735","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,24]]}}}