Water, Air, & Soil Pollution

, 228:415 | Cite as

Ecotoxicological Biomarkers in Multiple Tissues of the Neotenic Ambystoma spp. for a Non-lethal Monitoring of Contaminant Exposure in Wildlife and Captive Populations

  • C. Barriga-Vallejo
  • C. Aguilera
  • J. Cruz
  • J. Banda-Leal
  • D. Lazcano
  • R. MendozaEmail author


Amphibians are the most threatened vertebrate group with a third of currently known species endangered with extinction, as a result of climate change, habitat loss, disease-introduced exotic species, and pollution. Because of their vulnerability, they have often been used as environmental quality indicators, as well as laboratory models for toxicological research. Given the sensitivity of amphibians to changes in their surrounding environment, including pollution, it was deemed important to define a non-lethal technique based on the evaluation of a set of biomarkers in different tissues of neotenic individuals of Ambystoma velasci. The levels of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carboxylesterase (CaE), alkaline and acid phosphatases (ALP, ACP), glutathione s-transferase (GST), 7-ethoxyresorufin-O-deethylase (EROD), and superoxide dismutase (SOD) activities, as well as the oxygen radical absorption capacity (ORAC) were measured in tail, gills, liver, plasma, and brain samples. Significant tissue-specific differences were observed for all biomarkers with the exception of ACP. The highest values of specific activity for most biomarkers were detected in the liver. However, the levels measured in gills were very close to those observed in the liver and showed fewer variations than other tissues. These findings suggest that the sampling of gills could be used to evaluate pollution biomarkers in salamanders without apparent harm, as this tissue quickly regenerates.


Ambystoma spp. Esterases Detoxifying enzymes ORAC 



The authors wish to acknowledge the Program for the Support of Scientific and Technological Research, PAICYT CN356-15 for financing the project and CONACYT for the scholarship (#487242) given to one of the authors. Also to Sergio Luna, Eva Armijo, and Lucero Ortiz for their help to this project.


  1. Aguilera, C., González del Pliego, P., Mendoza, R., Lazcano, D., & Cruz, J. (2012). Pollution biomarkers in the spiny lizard (Sceloporus spp.) from two suburban populations of Monterrey, Mexico. Ecotoxicology (London, England), 21(8), 2103–2112. Scholar
  2. Aich, A., Goswami, A. R., Roy, U. S., & Mukhopadhyay, S. K. (2015). Ecotoxicological assessment of tannery effluent using guppy fish (Poecilia reticulata) as an experimental model: a biomarker study. Journal of Toxicology and Environmental Health, Part A, 78(4), 278–286. Scholar
  3. Al-Attar, A. M. (2004). The influence of dietary grapeseed oil on DMBA-induced liver enzymes disturbance in the frog, Rana ridibunda. Pakistan Journal of Nutrition, 3(5), 304–309. Scholar
  4. Attademo, A. M., Peltzer, P. M., Lajmanovich, R. C., Basso, A., & Junges, C. (2014). Tissue-specific variations of esterase activities in the tadpoles and adults of Pseudis paradoxa (anura: Hylidae). Water, Air, and Soil Pollution, 225(3):1903.
  5. Barriga-Vallejo, C., Hernandez-Gallegos, O., Von, I. H., López-Moreno, A. E., Ruiz-Gómez, M. d. L., Granados-Gonzalez, G., Garduño-Paz, M. V., Méndez-Sánchez, J., & Davis, A. K. (2015). Assessing population health of the Toluca axolotl Ambystoma rivulare (Taylor, 1940) from México, using leukocyte profiles. Herpetological Conservation and Biology, 10(2), 592–601.Google Scholar
  6. Barriga-Vallejo, C., Aguilera, C., Lazcano, D., & Mendoza, R. (2016). Biomarcadores en Ambystoma velasci de Galeana, Nuevo León (p. 123). Ciudad de México: Memorias en congreso: VII Congreso AMEQA.Google Scholar
  7. Bergeron, C. M., Bodinof, C. M., Unrine, J. M., & Hopkins, W. A. (2010). Mercury accumulation along a contamination gradient and nondestructive indices of bioaccumulation in amphibians. Environmental Toxicology and Chemistry, 29(4), 980–988. Scholar
  8. Bond, A. N. (1960). An analysis of the response of salamander gills to changes in the oxygen concentration of the medium. Developmental Biology, 20, 1–20. Scholar
  9. Bowerman, J., Johnson, P. T., & Bowerman, T. (2010). Sublethal predators and their injured prey: linking aquatic predators and severe limb abnormalities in amphibians. Ecology, 91(1), 242–251. Scholar
  10. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.CrossRefGoogle Scholar
  11. Brodeur, J. C., Suarez, R. P., Natale, G. S., Ronco, A. E., & Zaccagnini, M. E. (2011). Reduced body condition and enzymatic alterations in frogs inhabiting intensive crop production areas. Ecotoxicology and Environmental Safety, 74, 1370–1380. Scholar
  12. Burger, J., Campbell, K. R., Campbell, T. S., Shukla, T., Jeitner, C., & Gochfeld, M. (2005). Use of skin and blood as nonlethal indicators of heavy metal contamination in northern water snakes (Nerodia sipedon). Archives of Environmental Contamination and Toxicology, 49(2), 232–238. Scholar
  13. Burke, D. M., & Mayer, R. T. (1974). Ethoxyresorufin: direct fluorimetric assay of a microsomal O-dealkylation which is preferentially inducible by 3-methylcholanthrene. Drug Metabolism and Disposition, 2(February), 583–588.Google Scholar
  14. Chanson, J., Hoffmann, M., Cox, N., & Stuart, S. (2008). Threatened amphibians of the world. In S. N. Stuart, M. Hoffmann, J. S. Chanson, N. A. Cox, R. J. Berridge, P. Ramani, & B. E. Young (Eds.), The state of the world’s amphibians (pp. 33–52). Barcelona: Ingoprint S.A.Google Scholar
  15. Costa, M. J., Monteiro, D. A., Oliveira-Neto, A. L., Rantin, F. T., & Kalinin, A. L. (2008). Oxidative stress biomarkers and heart function in bullfrog tadpoles exposed to Roundup Original®. Ecotoxicology, 17(3), 153–163. Scholar
  16. Dabrowska, H., Kopko, O., Góra, A., Waszak, I., & Walkusz-Miotk, J. (2014). DNA damage, EROD activity, condition indices, and their linkages with contaminants in female flounder (Platichthys flesus) from the southern Baltic Sea. Science of the Total Environment, 496, 488–498. Scholar
  17. de Quiroga, G. B., Gutierrez, P., Rojo, S., & Alonso-Bedate, M. (1984). A comparative study of superoxide dismutase in amphibian tissues. Comparative Biochemistry and Physiology- Part B: Comparative Biochemistry, 77(3), 589–593.CrossRefGoogle Scholar
  18. Dennis, M. J., & Ort, C. A. (1977). The distribution of acetylcholine receptors on muscle fibers of regenerating salamander limbs. The Journal of Physiology, 266(3), 765–776.CrossRefGoogle Scholar
  19. Diario Oficial de la Federación (2010). NOM-059-SEMARNAT-2010. DOF 1–77. Mexico, D.F.Google Scholar
  20. Ellman, G. L., Courtney, K. D., Andres, V., & Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7(2), 88IN191-9095.CrossRefGoogle Scholar
  21. Ezemonye, L., & Tongo, I. (2010). Sublethal effects of endosulfan and diazinon pesticides on glutathione-S-transferase (GST) in various tissues of adult amphibians (Bufo regularis). Chemosphere, 81(2), 214–217. Scholar
  22. Frasco, M. F., & Guilhermino, L. (2002). Effects of dimethoate and beta-naphthoflavone on selected biomarkers of Poecilia reticulata. Fish Physiology and Biochemistry, 26(2), 149–156. Scholar
  23. Frías-Alvarez, P., Zúñiga-Vega, J. J., & Flores-Villela, O. (2010). A general assessment of the conservation status and decline trends of Mexican amphibians. Biodiversity and Conservation, 19(13), 3699–3742. Scholar
  24. Gorokhova, E., Löf, M., Reutgard, M., Lindström, M., & Sundelin, B. (2013). Exposure to contaminants exacerbates oxidative stress in amphipod Monoporeia affinis subjected to fluctuating hypoxia. Aquatic Toxicology, 127, 46–53. Scholar
  25. Goršič, M. (2007). Axolotl – a supermodel for tissue regeneration. Slovenian Veterinary Research, 44(1), 5–9.Google Scholar
  26. Gu, M., Ren, J., Sun, W., You, L., Yang, B., & Zhao, M. (2014). Isolation and identification of antioxidative peptides from frog (Hylarana guentheri) protein hydrolysate by consecutive chromatography and electrospray ionization mass spectrometry. Applied Biochemistry and Biotechnology, 173(5), 1169–1182. Scholar
  27. Henson-Ramsey, H., Kennedy-Stoskopf, S., Levine, J. F., Taylor, S. K., Shea, D., & Stoskopf, M. K. (2008). Acute toxicity and tissue distributions of malathion in Ambystoma tigrinum. Archives of Environmental Contamination and Toxicology, 55(3), 481–487. Scholar
  28. Homan, R. N., Regosin, J. V., Rodrigues, D. M., Reed, J. M., Windmiller, B. S., & Romero, L. M. (2003). Impacts of varying habitat quality on the physiological stress of spotted salamanders (Ambystoma maculatum). Animal Conservation, 6(1), 11–18. Scholar
  29. Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J. A., & Prior, R. L. (2002). High-throughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96-well format. Journal of Agricultural and Food Chemistry, 50(16), 4437–4444. Scholar
  30. Huang, T. L., Obih, P. O., Jaiswal, R., Hartley, W. R., & Thiyagarajah, A. (1997). Evaluation of liver and brain esterases in the spotted gar fish (Lepisosteus oculatus) as biomarkers of effect in the lower Mississippi River basin. Bulletin of Environmental Contamination and Toxicology, 58(5), 688–695. Scholar
  31. Huang, Y. W., Stegeman, J. J., Woodin, B. R., & Karasov, W. H. (2001). Immunohistochemical localization of cytochrome P450 1A induced by 3,3′, 4,4′, 5-pentachlorobiphenyl (PCB 126) in multiple organs of northern leopard frogs, Rana pipiens. Environmental Toxicology and Chemistry, 20(1), 191–197. Scholar
  32. Johnson, M. S., Vodela, J. K., Reddy, G., & Holladay, S. D. (2000). Fate and the biochemical effects of 2,4,6-trinitrotoluene exposure to tiger salamanders (Ambystoma tigrinum). Ecotoxicology and Environmental Safety, 46(2), 186–191. Scholar
  33. Johnson, P. T., Preu, E. R., Sutherland, D. R., Romansic, J. M., Han, B., & Blaustein, A. R. (2006). Adding infection to injury: synergistic effects of predation and parasitism on amphibian malformations. Ecology, 87(9), 2227–2235.CrossRefGoogle Scholar
  34. Kragl, M., Knapp, D., Nacu, E., Khattak, S., Maden, M., Epperlein, H. H., & Tanaka, E. M. (2009). Cells keep a memory of their tissue origin during axolotl limb regeneration. Nature, 460(7251), 60–65. Scholar
  35. Lajmanovich, R. C., Attademo, A. M., Simoniello, M. F., Poletta, G. L., Junges, C. M., Peltzer, P. M., et al. (2015). Harmful effects of the dermal intake of commercial formulations containing chlorpyrifos, 2, 4-D, and glyphosate on the common toad Rhinella arenarum (Anura: Bufonidae). Water, Air, & Soil Pollution, 226(12), 427. Scholar
  36. Mann, R. M., Hyne, R. V., Choung, C. B., & Wilson, S. P. (2009). Amphibians and agricultural chemicals: review of the risks in a complex environment. Environmental Pollution, 157(11), 2903–2927. Scholar
  37. Mazorra, M., Rubio, J., & Blasco, J. (2002). Acid and alkaline phosphatase activities in the clam Scrobicularia plana: kinetic characteristics and effects of heavy metals. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 131(2), 241–249. Scholar
  38. Moellmann, G., Lerner, A. B., & Hendee, J. R. (1974). The mechanism of frog skin lightening by acetylcholine. General and Comparative Endocrinology, 23(1), 45–51.CrossRefGoogle Scholar
  39. Mouchet, F., Gauthier, L., Mailhes, C., Ferrier, V., & Devaux, A. (2005). Comparative study of the comet assay and the micronucleus test in amphibian larvae (Xenopus laevis) using benzo(a)pyrene, ethyl methanesulfonate, and methyl methanesulfonate: establishment of a positive control in the amphibian comet assay. Environmental Toxicology, 20(1), 74–84. Scholar
  40. Nye, H. L. D., Cameron, J. A., Chernoff, E. a. G., & Stocum, D. L. (2003). Regeneration of the urodele limb: a review. Developmental Dynamics, 226(2), 280–294. Scholar
  41. Padidela, S., & Ravinder, R. T. (2015). Esterase variability in different tissues of flying frog (Rhacophorus lateralis) through polyacrylamide gelelectro phoresis. International Journal of Pharma Research & Review, 4(April), 7–12.Google Scholar
  42. Peskin, A. V., & Winterbourn, C. C. (2000). A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1). Clinica Chimica Acta, 293(1–2), 157–166. Scholar
  43. Pfleeger, A. Z., Eagles-Smith, C. A., Kowalski, B. M., Herring, G., Willacker, J. J., Jackson, A. K., & Pierce, J. R. (2016). From tails to toes: developing nonlethal tissue indicators of mercury exposure in five amphibian species. Ecotoxicology, 25(3), 574–583. Scholar
  44. Richardson, K. L., Castro, M. L., Schlenk, S. C., & Gardner, D. (2010). Polychlorinated biphenyls and biotransformation enzymes in three species of sea turtles from the Baja California peninsula of Mexico. Archives of Environmental Contamination and Toxicology, 58(1), 183–193. Scholar
  45. Rivera, M., & Davis, A. K. (2013). Evaluating a method for non-destructively obtaining small volumes of blood from gilled amphibians. Herpetological Review, 44(3), 428–430.Google Scholar
  46. Robles-Mendoza, C., Zúñiga-Lagunes, S. R., de León-Hill, C. A., Hernández-Soto, J., & Vanegas-Pérez, C. (2011). Esterases activity in the axolotl Ambystoma mexicanum exposed to chlorpyrifos and its implication to motor activity. Aquatic Toxicology, 105(3), 728–734. Scholar
  47. Sanchez-Hernandez, J. C. (2007). Ecotoxicological perspectives of β-esterases in the assessment of pesticide contamination. In R. H. Plattenberg (Ed.), Environmental pollution: new research (pp. 1–45). New York: Nova Science Publishers Inc..Google Scholar
  48. Scaps, P., Bernet, F., Gautron, J., & Boilly, B. (1994). Activities of acetylcholinesterase, choline acetyltransferase, and catecholamine production in the spinal cord of the axolotl Ambystoma mexicanum during forelimb regeneration. Biochemistry and Cell Biology, 72(5–6), 188–194.CrossRefGoogle Scholar
  49. Sparling, D. W., Linder, G., Bishop, C. A., & Krest, S. (Eds.). (2010). Ecotoxicology of amphibians and reptiles. Pensacola: CRC Press.Google Scholar
  50. Wake, D. B., & Vredenburg, V. T. (2008). Are we in the midst of the sixth mass extinction? A view from the world of amphibians. Proceedings of the National Academy of Sciences, 105(Supplement 1), 11466–11473. Scholar
  51. Wiklund, A. K. E., Adolfsson-Erici, M., Liewenborg, B., & Gorokhova, E. (2014). Sucralose induces biochemical responses in Daphnia magna. PLoS One, 9(4):e92771.
  52. Wilce, M. C. J., & Parker, M. W. (1994). Structure and function of glutathione S-transferases. Biochimica et Biophysica Acta, 1205, 1–18. Scholar
  53. Yora, T., & Sakagishi, Y. (1986). Comparative biochemical study of alkaline phosphatase isozymes in fish, amphibians, reptiles, birds and mammals. Comparative Biochemistry and Physiology: Part B: Comparative Biochemistry, 85(3), 649–658. Scholar
  54. Zambrano, L., Vega, E., Herrera, M. L. G., Prado, E., & Reynoso, V. H. (2007). A population matrix model and population viability analysis to predict the fate of endangered species in highly managed water systems. Animal Conservation, 10(3), 297–303. Scholar
  55. Zavala-Aguirre, J. L., Torres-Bugarin, O., & Zamora-Perez, A. L. (2007). Aquatic ecotoxicology approaches in Western Mexico. Journal of Environmental Science and Health. Part A, Toxic/hazardous Substances & Environmental Engineering, 42(10), 1503–1511. Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  • C. Barriga-Vallejo
    • 1
  • C. Aguilera
    • 1
  • J. Cruz
    • 2
  • J. Banda-Leal
    • 3
  • D. Lazcano
    • 3
  • R. Mendoza
    • 1
    • 4
    Email author
  1. 1.Facultad de Ciencias Biológicas, Laboratorio de EcofisiologíaUniversidad Autónoma de Nuevo León (UANL)San Nicolás de los GarzaMexico
  2. 2.Facultad de Medicina Veterinaria y ZootecniaUniversidad Autónoma de Nuevo León (UANL)EscobedoMexico
  3. 3.Facultad de Ciencias Biológicas, Laboratorio de HerpetologíaUniversidad Autónoma de Nuevo León (UANL)San Nicolás de los GarzaMexico
  4. 4.San Nicolás de los GarzaMexico

Personalised recommendations