Skip to main content
SpringerLink
Log in

Genotoxic Effects in Erythrocytes of Oreochromis niloticus Exposed to Nanograms-per-Liter Concentration of 17β-Estradiol (E2): An Assessment Using Micronucleus Test and Comet Assay

  • Published:
Water, Air, & Soil Pollution Aims and scope Submit manuscript

Abstract

Pharmacologically active substances used in the treatment of human and animal illnesses may usually enter the aquatic environment via effluents from sewage treatment plants, as they are not completely biodegraded or removed during waste water treatment. 17β-Estradiol genotoxicity was evaluated in Oreochromis niloticus (family Cichlidae) using micronucleus test, other nuclear abnormalities assessment, and the comet assay with erythrocytes. Fish were exposed to aqueous systems contaminated with 6 ng/L 17β-estradiol for three periods: 24 h, 48 h, and 10 days. The results showed that 17β-estradiol has genotoxic potential in different periods, since significant differences (P = 0.036) were observed in the micronucleus frequencies of the 10-day exposure groups in relation to the control group. Also, the same was observed when comparing the nuclear abnormality frequencies (P = 0.018) of the 24-h exposure group with the negative control group, and when using comet assay (P < 0.001) for 48-h evaluations. The tested concentration of the 17β-estradiol gave rise to mutagenic and genotoxic effects on the blood cells of O. niloticus, therefore the substance being considered a clastogenic chemical contaminant on both acute and chronic exposures. The assessment using a combination of micronucleus test, nuclear abnormalities, and comet assays proved to be suitable and useful in the genotoxicity testing of 17β-estradiol at nanograms per liter.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Ankley, G., Mihaich, E., Stahl, R., Tillitt, D., Colborn, T., McMaster, S., et al. (1998). Overview of a workshop on screening methods for detecting potential (anti-) estrogenic/androgenic chemicals in wildlife. Environmental Toxicology and Chemistry, 17(1), 68–67.

    Article  CAS  Google Scholar 

  • Ayllon, F., & Garcia-Vazquez, E. (2000). Induction of micronuclei and other nuclear abnormalities in European minnow phoxinus phoxinus and mollie poecilia latipinna: an assessment of the fish micronucleus test. Mutation Research, 467(2), 177–186.

    CAS  Google Scholar 

  • Belfroid, A. C., Van der Horst, A., Vethaak, A. D., Schafer, A. J., Rijs, G. B., Wegener, J., et al. (1999). Analysis and occurrence of estrogenic hormones and their glucuronides in surface water and waste water in The Netherlands. The Science of the Total Environment, 225(1–2), 101–108.

    Article  CAS  Google Scholar 

  • Belpaeme, K., Delbeke, K., Zhu, L., & Kirsch-Volders, M. (1996). Cytogenetic studies of PCB 77 on brown trut (Salmon truta fario) using the micronucleus test and the alkaline comet assay. Mutagenesis, 11, 485–492.

    Article  CAS  Google Scholar 

  • Birkett, J. W., & Lester, J. N. (2003). Endocrine disrupters in wastewater and sludge treatment process. Boca Raton: Lewis Publishers.

    Google Scholar 

  • Bonassi, S., Znaor, A., Ceppi, M., Lando, C., Chang, W. P., Holland, N., et al. (2007). An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans. Carcinogenesis, 28(3), 625–631.

    Article  CAS  Google Scholar 

  • Buschini, A., Martino, A., Gustavino, B., Monfrinotti, M., Poli, P., Rossi, C., et al. (2004). Cometa assay and micrnucleous test i circulatin eritrocites of Cyprinus carpio specimes expoused in situ to lake waters treated with desinfectants for potabilization. Mutation Research, 557, 119–129.

    CAS  Google Scholar 

  • Campana, M. A., Panzeri, A. M., Moreno, V. J., & Dulout, F. N. (1999). Genotoxic evaluation of the pyrethroid lambda-cyhalothrin using the micronucleus test in erythrocytes of the fish Cheirodon interruptus interruptus. Mutation Research, 438, 155–161.

    CAS  Google Scholar 

  • Carrasco, K. R., Tilbury, K. L., & Myers, M. S. (1990). Assessment of the piscine micronucleus test as in situ biological indicator of chemical contaminant effects. Canadian Journal of Fisheries and Aquatic Sciences, 47, 2123–2136.

    Article  CAS  Google Scholar 

  • Çavas, T., & Ergene-Gozukara, S. (2005a). Induction of micronuclei and nuclear abnormalities in oreochromis niloticus following exposure to petroleum refinery and chromium processing plant effluents. Aquatic Toxicology, 74(3), 264–271.

    Article  Google Scholar 

  • Çavas, T., & Ergene-Gozukara, S. (2003). Micronuclei, nuclear lesions and interphase silver-stained nucleolar organizer regions (AgNORs) as cyto-genotoxicity indicators in Oreochromis niloticus exposed to textile mill effluent. Mutation Research, 538(1–2), 81–91.

    Google Scholar 

  • Çavas, T., & Ergene-Gozukara, S. (2005b). Micronucleus test in fish cells: a bioassay for in situmonitoring of genotoxic pollution in the marine environment. Environmental and Molecular Mutagenesis, 46, 64–70.

    Article  Google Scholar 

  • Christensen, F. M. (1998). Pharmaceuticals in the environment—a human risk? Regulatory Toxicology and Pharmacology, 28(3), 212–221.

    Article  CAS  Google Scholar 

  • Collins, A. R. (2004). The comet assay for DNA damage and repair, principles, applications, and limitations. Molecular Biotechnology, 26, 249–261.

    Article  CAS  Google Scholar 

  • Collins, A. R., & Horvathova, E. (2001). Oxidative DNA damage, antioxidants and DNA repair: applications of the comet assay. Biochemical Society Transactions, 29(Pt 2), 337–341.

    Article  CAS  Google Scholar 

  • Colucci, M. S., Bork, H., & Topp, E. (2001). Persistence of estrogenic hormones in agricultural soils: I. 17Beta-estradiol and estrone. Journal of Environmental Quality, 30(6), 2070–2076.

    Article  CAS  Google Scholar 

  • Da Silva, J., Herrmann, S. M., Heuser, V., Peres, W., Possa Marroni, N., Gonzales-Gallego, J., et al. (2002). Evaluation of the genotoxic effect of rutin and quercetin by comet assay and micronucleus test. Food And Chemical Toxicology, 40, 941–947.

    Article  Google Scholar 

  • Desbrow, C., Routledge, E. J., Brighty, G. C., Sumpter, J. P., & Waldock, M. (1998). Identification of Estrogenic chemicals in STW Effluent. 1. Chemical fractionation and in vitro biologcal screening. Environmental Science & Technology, 32(11), 1549–1558.

    Article  CAS  Google Scholar 

  • Dorabawila, N., & Gupta, G. (2005). Endocrine disrupter–estradiol–in Chesapeake Bay tributaries. Journal of Hazardous Materials, 120(1–3), 67–61.

    Article  CAS  Google Scholar 

  • Fent, K., Weston, A. A., & Caminada, D. (2006). Ecotoxicology of human pharmaceuticals. Aquatic Toxicology, 76(2), 122–159.

    Article  CAS  Google Scholar 

  • Frenzilli, G., Bosco, E., & Barale, R. (2000). Validation of single cell gel assay in human leukocytes with 18 reference compounds. Mutation Research, 468(2), 93–108.

    CAS  Google Scholar 

  • Frenzilli, G., Nigro, M., & Lyons, B. P. (2009). The Comet assay for the evaluation of genotoxic impact in aquatic environments. Mutation Research, 681(1), 80–92.

    Article  CAS  Google Scholar 

  • Ghiselli, G., & Jardim, W. F. (2007). Interferentes endócrinos no ambiente. Quimica Nova, 30(3), 695–606.

    Article  Google Scholar 

  • Gillesby, B. E., & Zacharewski, T. R. (1998). Exoestrogens: mechanisms of action and strategies for identification and assessment. Environmental Toxicology and Chemistry, 17(1), 3–14.

    Article  CAS  Google Scholar 

  • Goode, E. L., Ulrich, C. M., & Potter, J. D. (2002). Polymorphisms in DNA repair genes and associations with cancer risk. Cancer Epidemiology, Biomarkers & Prevention, 11(12), 1513–1530.

    CAS  Google Scholar 

  • Grisolia, C. K., & Starling, F. L. (2001). Micronuclei monitoring of fishes from Lake Paranoa, under influence of sewage treatment plant discharges. Mutation Research, 491(1–2), 39–44.

    CAS  Google Scholar 

  • Guilherme, S., Valega, M., Pereira, M. E., Santos, M. A., & Pacheco, M. (2007). Erythrocytic nuclear abnormalities in wild and caged fish (Liza aurata) along an environmental mercury contamination gradient. Ecotoxicology and Environmental Safety, 70(3), 411–412.

    Article  Google Scholar 

  • Gustavino, B., Scornajenghi, K. A., Minissi, S., & Ciccotti, E. (2001). Micronuclei induced in erythrocytes of cyprinus carpio (teleostei, pisces) by X-rays and colchicine. Mutation Research, 494(1–2), 151–159.

    CAS  Google Scholar 

  • Heddle, J. A. (1973). A rapid in vivo test for chromosomal damage. Mutation Research, 18(2), 187–190.

    CAS  Google Scholar 

  • Heddle, J. A., Cimino, M. C., Hayashi, M., Romagna, F., Shelby, M. D., Tucker, J. D., et al. (1991). Micronuclei as an index of cytogenetic damage: past, present, and future. Environmental and Molecular Mutagenesis, 18(4), 277–291.

    Article  CAS  Google Scholar 

  • Hoshina, M. M., de Franceschi de Angelis, D., & Marin-Morales, M. A. (2008). Induction of micronucleus and nuclear alterations in fish (Oreochromis niloticus) by a petroleum refinery effluent. Mutation Research, 656(1–2), 44–48.

    CAS  Google Scholar 

  • Hughes, C. (2007). Are the differences between estradiol and other estrogens merely semantical ? The Journal of Clinical Endocrinology and Metabolism, 81(6), 2405.

    Article  Google Scholar 

  • Hundal, B. S., Dhillon, V. S., & Sidhu, I. S. (1997). Genotoxic potential of estrogens. Mutation Research, 389(2–3), 173–181.

    CAS  Google Scholar 

  • Joosten, H. F., van Acker, F. A., van den Dobbelsteen, D. J., Horbach, G. J., & Krajnc, E. I. (2004). Genotoxicity of hormonal steroids. Toxicology Letters, 151(1), 113–134.

    Article  CAS  Google Scholar 

  • Kammann, U., Bunke, M., Steinhart, H., & Theobald, N. (2001). A permanent fish cell line (EPC) for genotoxicity testing of marine sediments with the comet assay. Mutation Research, 498(1–2), 67–77.

    CAS  Google Scholar 

  • Koppen, G., Toncelli, L. M., Triest, L., & Verschaeve, L. (1999). The comet assay: a tool to study alteration of DNA integrity in developing plant leaves. Mechanisms of Ageing and Development, 110(1–2), 13–24.

    Article  CAS  Google Scholar 

  • Kummerer, K. (2000). Drugs, diagnostic agents and disinfectants in wastewater and water–a review. Schriftenreihe des Vereins für Wasser-, Boden- und Lufthygiene, 105, 59–71.

    CAS  Google Scholar 

  • Lemos, N. G., Dias, A. L., Silva-Souza, A. T., & Mantovani, M. S. (2005). Evaluation of environmental waters using the comet assay in Tilapia rendalli. Environmental Toxicology and Pharmacology, 19, 197–101.

    Article  CAS  Google Scholar 

  • Liehr, J. G. (1990). Genotoxic effects of estrogens. Mutation Research, 238(3), 269–276.

    CAS  Google Scholar 

  • Lintelmann, J., Katayama, A., Kurihara, N., Shore, L., & Wenzel, A. (2003). Endocrine disruptors in the environmet. Pure and Applied Chemistry, 75(5), 631–681.

    Article  CAS  Google Scholar 

  • López de Alda, M., & Barceló, D. (2001). Determination of steroid sex hormones and related synthetic compounds considered as endocrine disrupters in water by fully automated on-line solid-phase extraction-liquid chromatography-diode array detectioon. Journal of Chromatography A, 911, 203–210.

    Article  Google Scholar 

  • Matsumoto, S. T., Mantovani, M. S., Malaguttii, M. I., Dias, A. L., Fonseca, I. C., & Marin-Morales, M. A. (2006). Genotoxicity and mutagenicity of water contaminated with tanney efluents, as evaluated by the micronucleus test and comet assay using the fish Oreocromis niloticus and chromossome aberrations in onion root-tips. Genetics And Molecular Biology, 29(1), 148–158.

    Article  CAS  Google Scholar 

  • Meade-Callahan, M. J. (2007). Los Microbios: Cómo Funcionan y Cómo los Cambian los Antibióticos. http://www.actionbioscience.org. Accessed 30 Apr 2010.

  • Moron, S., Polez, V., Artoni, R., Ribas, J., & Takahashi, H. (2006). Estudo de alterações na concentração dos íons plasmátcos e da indução de micronúcleos em Piraructus mesopotamicus esposto ao herbicida atrazina. Journal of the Brazilian Society of Ecotoxicology, 1(1), 27–30.

    Article  Google Scholar 

  • Pacheco, M., & Santos, M. A. (1998). Induction of liver EROD and erythrocytic nuclear abnormalities by cyclophosphamide and PAHs in Anguilla anguilla L. Ecotoxicology and Environmental Safety, 40(1–2), 71–76.

    Article  CAS  Google Scholar 

  • Russo, C., Lucia, R., Morescalchi, M. A., & Stingo, V. (2004). Assessement of environmental stress by the micronucleus test and the comet assay on the genome of teleost population from to natural environments. Ecotoxicology and Environmental Safety, 57, 168–174.

    Article  CAS  Google Scholar 

  • Sánchez-Galán, S., Linde, A. R., Izquierdo, J., & García-Vásquez, E. (1998). Micronuclei and fluctuating asymmetry in brown trout (Salmo trutta): complementary methods to biomonitor freshwater ecosystens. Mutation Research, 412, 219–225.

    Google Scholar 

  • Schmezer, P., Rajaee-Behbahani, N., Risch, A., Thiel, S., Rittgen, W., Drings, P., et al. (2001). Rapid screening assay for mutagen sensitivity and DNA repair capacity in human peripheral blood lymphocytes. Mutagen, 16(1), 25–30.

    Article  CAS  Google Scholar 

  • Schmid, W. (1975). The micronucleus test. Mutation Research, 31(1), 9–15.

    CAS  Google Scholar 

  • Singh, N. P., McCoy, M. T., Tice, R. R., & Schneider, E. L. (1988). A simple technique for quantization of low levels of DNA damage in individual cells. Experimental Cell Research, 175(1), 184–191.

    Article  CAS  Google Scholar 

  • Solé, M., Raldua, D., Barceló, D., & Porte, C. (2003). Long term exposure effects in vetellogenin, sex hormones, and biotransformation enzymes in female carp in relation to a sewage treatment works. Ecotoxicology and Environmental Safety, 56, 373–380.

    Article  Google Scholar 

  • Strunjak-Perovic, I., Coz-Rakovac, R., Popovic, A. T., & Jadan, M. (2008). Seansonality of nuclear abnormalities in gilthead sea bream Sparus aurata (L.) erytrocytes. Fish Physiology and Biochemistry, 35(2), 287–291.

    Article  Google Scholar 

  • Teles, M., Pacheco, M., & Santos, M. A. (2006). Biotransformation, stress and genotoxic effects of 17beta-estradiol in juvenile sea bass (Dicentrarchus labrax L.). Environment International, 32(4), 470–477.

    Article  CAS  Google Scholar 

  • Ternes, T. A., Stumpf, M., Mueller, J., Haberer, K., Wilken, R. D., & Servos, M. (1999). Behavior and occurrence of estrogens in municipal sewage treatment plants--I. Investigations in Germany, Canada and Brazil. Science of The Total Environment, 225(1-2), 81–80.

    Article  CAS  Google Scholar 

  • Ventura, B. C., Angelis, D. F., & Marin-Morales, M. A. (2008). Mutagenic and genotoxic effects of the Atrazine herbicide in Oreochromis niloticus (Perciformes, Cichlidae) detected by the micronuclei test and the comet assay. Pesticide Biochemistry And Physiology, 90, 42–51.

    Article  CAS  Google Scholar 

  • WHO. (1993). Biomarkers and risk assessment: concepts and principles. IPCS, 155. Geneva: WHO.

    Google Scholar 

  • Yared, E., McMillan, T. J., & Martin, F. L. (2002). Genotoxic effects of oestrogens in breast cells detected by the micronucleus assay and the comet assay. Mutagen, 17(4), 345–352.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to thank the Universidade Positivo for the financial support, as well as Luciana Requião and Fernando Zasso for their technical assistance.

Conflict of Interest Statement

The authors declare that there are no conflicts of interest.

Author information

Authors and Affiliations

  1. Mestrado Profissional em Gestão Ambiental, Universidade Positivo, Prof. Pedro Viriato Parigot de Souza Street, 5300, 81280-330, Curitiba, Paraná, Brazil

    Graziela Sponchiado, Eliana Mara Fortunato de Lucena Reynaldo, Eliane Carvalho de Vasconcelos & Cíntia Mara Ribas de Oliveira

  2. Grupo de Pesquisa em Farmacologia e Fisiopatologia da pele, Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Paraná, UFPR, Curitiba, Paraná, Brazil

    Graziela Sponchiado

  3. Núcleo de Ciências Biológicas e da Saúde, Curso de Farmácia, Universidade Positivo, Curitiba, Paraná, Brazil

    Any Caroline B. de Andrade, Eliane Carvalho de Vasconcelos & Cíntia Mara Ribas de Oliveira

  4. Núcleo de Biologia, Centro Acadêmico de Vitória de Santo Antão:UFPE, Vitória de Santo Antão, Pernambuco, Brazil

    Mônica Lucia Adam

  5. Programa de Pós-Graduação em Ciências Biológicas, Centro de Ciências Biológicas, UFPE, Recife, Pernambuco, Brazil

    Mônica Lucia Adam

Authors
  1. Graziela Sponchiado
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eliana Mara Fortunato de Lucena Reynaldo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Any Caroline B. de Andrade
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eliane Carvalho de Vasconcelos
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mônica Lucia Adam
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Cíntia Mara Ribas de Oliveira
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Cíntia Mara Ribas de Oliveira.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sponchiado, G., de Lucena Reynaldo, E.M.F., de Andrade, A.C.B. et al. Genotoxic Effects in Erythrocytes of Oreochromis niloticus Exposed to Nanograms-per-Liter Concentration of 17β-Estradiol (E2): An Assessment Using Micronucleus Test and Comet Assay. Water Air Soil Pollut 218, 353–360 (2011). https://doi.org/10.1007/s11270-010-0649-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11270-010-0649-9

Keywords

Search

Navigation