Cytotoxic and genotoxic effects induced by enrofloxacin-based antibiotic formulation Floxagen® in two experimental models of bovine cells in vitro: peripheral lymphocytes and cumulus cells

  • Juan Patricio Anchordoquy
  • Juan Mateo Anchordoquy
  • Noelia Nikoloff
  • Rocío Gambaro
  • Gisel Padula
  • Cecilia Furnus
  • Analía Seoane
Research Article


The in vitro effect of enrofloxacin (EFZ) was tested on two experimental somatic bovine cells in vitro: peripheral lymphocytes (PLs) and cumulus cells (CCs). The cytotoxicity and genotoxicity of this veterinary antibiotic were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, single-cell gel electrophoresis (SCGE) assay, and cytokinesis-block micronucleus cytome (CBMN cyt) assay. Cells were treated during 24 h, and three concentrations were tested (50 μg/mL, 100 μg/mL, 150 μg/mL). When EFZ was tested in PLs, the results demonstrated that the antibiotic was able to induce cell death and DNA damage with all concentrations. In addition, 50 μg/mL and 100 μg/mL EFZ increased frequencies of micronuclei (MNi). On the other hand, the highest EFZ concentration occasioned cellular cytotoxicity in CCs as evidenced by mitochondrial activity alterations. Nevertheless, EFZ was not able to induce DNA damage and MNi in CCs. These results represent the first experimental evidence of genotoxic and cytotoxic effects exerted by EFZ in bovine PLs and CCs.


Bovine cells MTT SCGE assay CBMN cyt assay Enrofloxacin 



We are grateful to the staff of SENASA from Frigorífico Gorina S.A. for providing the bovine ovaries.


This work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica de la República Argentina (PICT BID 1972-2013); Ministerio de Ciencia, Tecnología e Innovación Productiva de la Nación Argentina; Consejo Nacional de Investigaciones Científicas y Tecnológicas (PIP 112-20130100657); and Universidad Nacional de La Plata (V246 and V249).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. Altreuther P (1987) Data on chemistry and toxicology of Baytril. Vet Med Rev 2:87–89Google Scholar
  2. Anchordoquy JM, Anchordoquy JP, Nikoloff N, Pascua AM, Furnus CC (2017) High copper concentrations produce genotoxicity and cytotoxicity in bovine cumulus cells. Environ Sci Pollut Res Int 24(24):20041–20049CrossRefGoogle Scholar
  3. Araldi RP, de Melo TC, Mendes TB, de Sá Júnior PL, Nozima BH, Ito ET, de Carvalho RF, de Souza EB, de Cassia Stocco R (2015) Using the comet and micronucleus assays for genotoxicity studies: a review. Biomed Pharmacother 72:74–82CrossRefGoogle Scholar
  4. Ayaki M, Atsuo I, Mitsutaka S, Shigeo Y, Ryohei K (2010) Cytotoxicity of five fluoroquinolone and two nonsteroidal anti-inflammatory benzalkonium chloride-free ophthalmic solutions in four corneoconjunctival cell lines. Clin Ophthalmol 4:1019–1024CrossRefGoogle Scholar
  5. Babaei H, Roshangar L, Sakhaee E, Abshenas J, Kheirandish R, Dehghani R (2012) Ultrastructural and morphometrical changes of mice ovaries following experimentally induced copper poisoning. Iran Red Crescent Med J 14(9):558–568Google Scholar
  6. Bautista Garfias CR, Acosta García E, Toledo GI (2000) Evaluación del bioensayo de MTT para determinar la proliferación in vitro de linfocitos de bovino, frescos y congelados Vet. Méx 31(2)Google Scholar
  7. Boxall ABA (2010) Veterinary medicines and the environment. Comparative and veterinary pharmacology. Handb Exp Pharmacol 199:291–313CrossRefGoogle Scholar
  8. Brown SA (1996) Fluoroquinolones in animal health. J Vet Pharmacol Therap 19:1–14CrossRefGoogle Scholar
  9. Ceko MJ, Hummitzsch K, Hatzirodos N, Rodgers RJ, Harris HH (2015) Quantitative elemental analysis of bovine ovarian follicles using X-ray fluorescence imaging. Metallomics 7(5):828–836CrossRefGoogle Scholar
  10. Chen T, Yuana J, Feng X, Wei H, Hua W (2011) Effects of enrofloxacin on the human intestinal microbiota in vitro. Int J Antimicrob Agents 37:567–571CrossRefGoogle Scholar
  11. Collins AR (2004) The comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol 26:249–261CrossRefGoogle Scholar
  12. Corn CM, Hauser-Kronberger C, Moser M, Tews G, Ebner T (2005) Predictive value of cumulus cell apoptosis with regard to blastocyst development of corresponding gametes. Fertil Steril 84(3):627–633CrossRefGoogle Scholar
  13. Dutra A, Pak E, Wincovitch S, John K, Poirier MC, Olivero OA (2010) Nuclear bud formation: a novel manifestation of zidovudine genotoxicity. Cytogenet Genom Res 128:105–110CrossRefGoogle Scholar
  14. Fenech M (2007) Cytokinesis-block micronucleus cytome assay. Nat Protoc 2:1084–1104CrossRefGoogle Scholar
  15. Fenech M, Kirsch-Volders M, Natarajan AT (2011) Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis 26(1):125–132CrossRefGoogle Scholar
  16. Gorla N, Ovando G, Larripa I (1999) Chromosomal aberrations in human lymphocytes exposed in vitro to enrofloxacin and ciprofloxacin. Toxicol Lett 104:43–48CrossRefGoogle Scholar
  17. Botelho RG, Monteiro SH, Tornisielo VL (2015) Veterinary antibiotics in the environment. Chapter 5. IntechGoogle Scholar
  18. Halling-Sørensen B, Nors Nielsen S, Lanzky PF, Ingerslev F, Holten Lützhøft HC, Jørgensen SE (1998) Occurrence, fate and effects of pharmaceutical substances in the environment—a review. Chemosphere 36:357–393CrossRefGoogle Scholar
  19. Hamscher G, Sczesny S, Höper H, Nau H (2002) Determination of persistent tetracycline residues in soil fertilized with liquid manure by high performance liquid chromatography with electrospray ionization tandem mass spectrometry. Anal Chem 74:1509–1518CrossRefGoogle Scholar
  20. Hartmann A, Alder AC, Koller T, Widmer RM (1998) Identification of fluoroquinolone antibiotics as the main source of umuC genotoxicity in native hospital wastewater. Environ Toxicol Chem 17(3):377–382CrossRefGoogle Scholar
  21. Hooper DC, Wolfson J (1991) Mode of action of the new quinolones: new data. Eur J Clin Microbiol Infect Dis 10:223–231CrossRefGoogle Scholar
  22. Høst E, Gabrielsen A, Lindenberg S, Smidt-Jensen S (2002) Apoptosis in human cumulus cells in relation to zona pellucida thickness variation, maturation stage, and cleavage of the corresponding oocyte after intracytoplasmic sperm injection. Fertil Steril 77(3):511–515CrossRefGoogle Scholar
  23. Kołodziejska M, Maszkowska J, Bialk-Bielin A, Steudte S, Kumirska J, Stepnowski P, Stolte S (2013) Aquatic toxicity of four veterinary drugs commonly applied in fish farming and animal husbandry. Chemosphere 92:1253–1259CrossRefGoogle Scholar
  24. Krisher RL (2004) The effect of oocyte quality on development. J Anim Sci 82:14–23Google Scholar
  25. Kϋmmerer K, Al-Ahmad A, Mersch-Sundermann V (2000) Biodegradability of some antibiotics, elimination of the genotoxicity and affection of wastewater bacteria in a simple test. Chemosphere 40:701–710CrossRefGoogle Scholar
  26. Liu B, Cui Y, Brown PB, Ge X, Xie J, Xu P (2015) Cytotoxic effects and apoptosis induction of enrofloxacin in hepatic cell line of grass carp (Ctenopharyngodon idellus). Fish Shellfish Immunol 47:639–644CrossRefGoogle Scholar
  27. Madhuresh KS, Rusha G, Rohit S, Sanjay M, Purbita C, Anil T (2016) Genotoxic impurities evaluation in active pharmaceutical ingredients (API)/drug substance. Pharm Lett 8(12):234–243Google Scholar
  28. Magdaleno A, Carusso S, Moretton J (2017) Toxicity and genotoxicity of three antimicrobials commonly used in veterinary medicine. Bull Environ Contam Toxicol, In press 99:315–320CrossRefGoogle Scholar
  29. Olive PL (1999) DNA damage and repair in individual cells: applications of the comet assay in radiobiology. Int J Radiat Biol 75:395–405CrossRefGoogle Scholar
  30. Otero JL, Mestorino N, Errecalde JO (2001) Enrofloxacina una fluoroquinolona de uso exclusive en veterinaria pate II: farmacocinética y toxicidad. Analecta Veterinaria 21(1):42–49Google Scholar
  31. Pavanello S, Levis AG (1994) Human peripheral blood lymphocytes as a cell model to evaluate the genotoxic effect of coal tar treatment. Environ Health Perspect 102(9):95–99CrossRefGoogle Scholar
  32. Prescott JF, Baggot JD, Walker RD (2000) Antimicrobial therapy in veterinary medicine. Iowa State University Press, AmesGoogle Scholar
  33. Radko L, Minta M, Stypuła-Trębas S (2013) Influence of fluoroquinolones on viability of Balb/c 3T3 and HepG2 cells. Bull Vet Inst Pulawy 57(4):599–606CrossRefGoogle Scholar
  34. Robb J, Norval M, Neill WA (1990) The use of tissue culture for the detection of mycotoxins. Lett Appl Microbiol 10:161–165CrossRefGoogle Scholar
  35. Santos RR, Schoevers EJ, Roelen BA (2014) Usefulness of bovine and porcine IVM/IVF models for reproductive toxicology. Reprod Biol Endocrinol: RB&E 12:117. CrossRefGoogle Scholar
  36. Seino T, Saito H, Kaneko T, Takahashi T, Kawachiya S, Kurachi H (2002) Eight-hydroxy-2′-deoxyguanosine in granulosa cells is correlated with the quality of oocytes and embryos in an in vitro fertilization-embryo transfer program. Fertil Steril 77(6):1184–1190CrossRefGoogle Scholar
  37. Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191CrossRefGoogle Scholar
  38. Tanghe S, Van Soom A, Nauwynck H, Coryn M, de Kruif A (2002) Minireview: Functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization. Mol Reprod Dev 61:414–424CrossRefGoogle Scholar
  39. Tice R, Strauss GH (1995) The single cell gel electrophoresis/comet assay: a potential tool for detecting radiation-induced DNA damage in humans. Stem Cells 13(1):207–214Google Scholar
  40. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35(3):206–221CrossRefGoogle Scholar
  41. Trouchon T, Lefebvre S (2016) A review of enrofloxacin for veterinary use. Open J Vet Med 6:40–58CrossRefGoogle Scholar
  42. Tsai TH, Chen WL, Hu FR (2010) Comparison of fluoroquinolones: cytotoxicity on human corneal epithelial cells. Eye 24:909–917CrossRefGoogle Scholar
  43. Vasquez MZ (2010) Combining the in vivo comet and micronucleus assays: a practical approach to genotoxicity testing and data interpretation. Mutagenesis 25(2):187–199CrossRefGoogle Scholar
  44. Wu J, Tu D, Yuan L-Y, Yuan H, Wen L-X (2013) T-2 toxin exposure induces apoptosis in rat ovarian granulosa cells through oxidative stress. Environ Toxicol Pharmacol 36:493–500CrossRefGoogle Scholar
  45. Yan H, Tian M, Row KH (2008) Determination of enrofloxacin and ciprofloxacin in milk using molecularly imprinted solid-phase extraction. J Sep Sci 31:3015–3020CrossRefGoogle Scholar
  46. Yu F, Yu S, Yu L, Li Y, Wu Y, Zhang H, Qu L, Harrington PB (2014) Determination of residual enrofloxacin in food samples by a sensitive method of chemiluminescence enzyme immunoassay. Food Chem 149:71–75CrossRefGoogle Scholar
  47. Yuan YQ, Van Soom A, Leroy JL, Dewulf J, Van Zeveren A, de Kruif A, Peelman LJ (2005) Apoptosis in cumulus cells but not in oocytes may influence bovine embryonic developmental competence. Theriogenology 63:2147–2163CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.IGEVET, Instituto de Genética Veterinaria “Ing. Fernando N Dulout” (UNLP-CONICET-CONICET LA PLATA), Facultad de Ciencias Veterinarias – UNLPUniversidad Nacional de La Plata - CONICETLa PlataArgentina
  2. 2.Facultad de Ciencias Naturales y Museo – UNLPLa PlataArgentina
  3. 3.Cátedra de Citología, Histología y Embriología “A”Facultad de Ciencias Médicas – UNLPLa PlataArgentina

Personalised recommendations