, Volume 20, Issue 6, pp 1315–1327 | Cite as

Antioxidant response and metal accumulation in tissues of Iberian green frogs (Pelophylax perezi) inhabiting a deactivated uranium mine

  • Sérgio M. MarquesEmail author
  • Sara C. Antunes
  • Bruno Nunes
  • Fernando Gonçalves
  • Ruth Pereira


Human mining activities tend often to generate greatly impacted areas which remain contaminated for long periods of time, giving rise to extreme habitats. Mining sites are usually characterized for the production of metal rich effluents with very low pH. In this work we analyzed physical and chemical parameters of water from a deactivated uranium mine pond (M) and a reference site (REF) as well as their metal content. Furthermore, we determined and compared metal accumulation in liver, kidney, bones, muscle and skin of Pelophylax perezi from REF with P. perezi from M. We also determined the enzymatic activities of glutathione-S-transferases (GSTs), catalase (CAT), glutathione reductase (Gred), and glutathione peroxidase (GPx; both selenium-dependent and selenium-independent) in liver, kidney, lung and heart. Additionally, lipoperoxidation (LPO) was also assessed in the same tissues via thiobarbituric acid reactive substances (TBARS) assay and lactate dehydrogenase (LDH) activity was determined in muscle. Our results revealed that the majority of metals were in higher concentrations in tissues of organisms from M. This trend was especially evident for U whose content reached a difference of 1350 fold between REF and M organisms. None of the organs tested for antioxidant defenses revealed LPO, nonetheless, with exception for liver, all organs from the M frogs presented increased total GPx activity and selenium-dependent GPx. However, this response was significant only for the lung, probably as a consequence of the significant inhibition of CAT upstream and to cope with the subsequent increase in H2O2. Lungs were the organs displaying greater responsiveness of the anti-oxidant stress system in frogs from the uranium mine area.


Oxidative stress Biomarkers Uranium mine Pelophylax perezi Metals 



Authors wish to acknowledge EDM for their collaboration. Sérgio M. Marques was supported by a PhD grant (ref. SFRH/BD/38282/2007) and Sara C. Antunes was recipient of a post-doctoral fellowship (ref. SFRH/BPD/40052/2007) from Fundação para a Ciência e Tecnologia (Portuguese Ministry of Science, Technology and Higher Education). This research is part of the projects Engenur (ref. PTDC/AAC-AMB/114057/2009) and UraniumRisk (ref. POCI/AMB/60899/2004) funded by the Portuguese Government (Program Ciência - Inovação 2010) and by the European Social Fund. This research was also partially funded by FSE and POPH funds (Programa Ciência 2007).


  1. Aebi H (1984) Catalase in vitro. Method Enzymol 105:121–126. doi: 10.1016/S0076-6879(84)05016-3 CrossRefGoogle Scholar
  2. André A, Antunes SC, Gonçalves F, Pereira R (2009) Bait-lamina assay as a tool to assess the effects of metal contamination in the feeding activity of soil invertebrates within a uranium mine area. Environ Pollut 157(8–9):2368–2377. doi: 10.1016/j.envpol.2009.03.023 CrossRefGoogle Scholar
  3. Antunes SC, de Figueiredo DR, Marques SM, Castro BB, Pereira R, Gonçalves F (2007a) Evaluation of water column and sediment toxicity from an abandoned uranium mine using a battery of bioassays. Sci Total Environ 374:252–259. doi: 10.1016/j.scitotenv.2006.11.025 CrossRefGoogle Scholar
  4. Antunes SC, Pereira R, Gonçalves F (2007b) Acute and chronic toxicity of effluent water from an abandoned uranium mine. Arch Environ Contam Toxicol 53(2):207–213. doi: 10.1007/s00244-006-0011-9 CrossRefGoogle Scholar
  5. Antunes SC, Pereira R, Gonçalves F (2007c) Evaluation of the potential toxicity (acute and chronic) of sediments from an abandoned uranium mine ponds. J Soil Sediment 7(6):368–376. doi: 10.1065/jss2007.08.247 CrossRefGoogle Scholar
  6. Antunes SC, Castro BB, Pereira R, Gonçalves F (2008) Contribution for tier 1 of the ecological risk assessment of Cunha Baixa uranium mine (central Portugal): II. Soil ecotoxicological screening. Sci Total Environ 390:387–395. doi: 10.1016/j.scitotenv.2007.07.053 CrossRefGoogle Scholar
  7. APHA (1995) Standard methods for the examination of water and wastewater, 19th edn. ASTM, Washington, DCGoogle Scholar
  8. Arruda-Neto JD, Guevara MV, Nogueira GP, Saiki M, Cestari AC, Shtejer K, Deppman A, Filho JWP, Garcia F, Geraldo LP, Gouveia AN, Guzmán F, Mesa J, Rodriguez O, Semmler R, Vanin VR (2004) Long-term accumulation of uranium in bones of Wistar rats as function of intake dosages. Radiat Prot Dosim 112(3):385–393. doi: 10.1093/rpd/nch405 CrossRefGoogle Scholar
  9. Atli G, Alptekin O, Tükel S, Canli M (2006) Response of catalase activity of Ag+, Cd2+, Cr6+, Cu2+ and Zn2+ in five tissues of freshwater Oreochromis niloticus. Comp Biochem Physiol 143C:218–224. doi: 10.1016/j.cbpc.2006.02.003 Google Scholar
  10. ATSDR—Agency for Toxic Substances and Disease Registry (1999) Toxicological profile for uranium. U.S. Department of health and human services, Atlanta, GAGoogle Scholar
  11. ATSDR—Agency for Toxic Substances and Disease Registry (2004) Toxicological profile for cobalt. U.S. Department of health and human services, Atlanta, GAGoogle Scholar
  12. ATSDR—Agency for Toxic Substances and Disease Registry (2006) Toxicological profile for aluminum. U.S. Department of health and human services, Atlanta, GAGoogle Scholar
  13. Barillet S, Adam C, Palluel O, Devaux A (2007) Bioaccumulation, oxidative stress, and neurotoxicity in Danio rerio exposed to different isotopic compositions of uranium. Environ Toxicol Chem 26(3):497–505. doi: 10.1897/06-243R.1 CrossRefGoogle Scholar
  14. Behne D, Wolters W (1983) Distribution of selenium and glutathione peroxidase in the rat. J Nutr 113:456–461Google Scholar
  15. Bondy SC, Ali SF, Guo-Ross S (1998) Aluminum but not iron induces pro-oxidant events in the rat brain. Mol Chem Neuropathol 34(2–3):219–232. doi: 10.1007/BF02815081 CrossRefGoogle Scholar
  16. Bozhkov A, Padalko V, Dlubovskaya V, Menzianova N (2010) Resistance to heavy metal toxicity in organisms under chronic exposure. Indian J Exp Biol 48:679–696Google Scholar
  17. Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254. doi: 10.1016/0003-2697(76)90527-3 CrossRefGoogle Scholar
  18. Buege JA, Aust SD (1978) Microssomal lipid peroxidation. Method Enzymol 52:302–310. doi: 10.1016/S0076-6879(78)52032-6 CrossRefGoogle Scholar
  19. Carlberg I, Mannervik B (1985) Glutathione reductase. Methods Enzymol 113:484–490. doi: 10.1016/S0076-6879(85)13062-4 CrossRefGoogle Scholar
  20. Cooley HM, Klaverkamp JF (2000) Accumulation and distribution of dietary uranium in Lake Whitefish (Coregonus clupeaformis). Aquat Toxicol 48:477–494. doi: 10.1016/S0166-445X(99)00058-2 CrossRefGoogle Scholar
  21. Cooper S, Fortin C (2010) Metal and metallothionein content in bullfrogs: study of a whole watershed impacted by agricultural activities. Ecotox Environ Safe 73(3):391–399. doi: 10.1016/j.ecoenv.2009.12.006 CrossRefGoogle Scholar
  22. Drolet-Vives K, Zayed J, Sauvé S (2009) Assessment of hair and bone accumulation of beryllium by mice exposed to contaminated dusts. J Appl Toxicol 29(7):638–642. doi: 10.1002/jat.144 CrossRefGoogle Scholar
  23. Farombi EO, Adelowo OA, Ajimoko YR (2007) Biomarkers of oxidative stress and heavy metal levels as indicators of environmental pollution in African cat fish (Clarias gariepinus) from Nigeria Ogun River. Int J Environ Res Public Health 4(2):158–165. doi: 10.3390/ijerph2007040011 CrossRefGoogle Scholar
  24. Fisenne IM (1994) Uranium. In: Seiler HG, Sigel A, Sigel H (eds) Handbook on metals in clinical and analytical chemistry. Marcel Dekker, Inc, New YorkGoogle Scholar
  25. Flohé L, Günzler WA (1984) Assays of glutathione peroxidase. Method Enzymol 105:114–120CrossRefGoogle Scholar
  26. Franco R, Sánchez-Olea R, Reyes-Reyes EM, Panayiotidis M (2009) Environmental toxicity, oxidative stress and apoptosis: Ménage à Trois. Mutat Res 674:3–22. doi: 10.1016/j.mrgentox.2008.11.012 Google Scholar
  27. Galaris D, Evangelou A (2002) The role of oxidative stress in mechanism of metal-induced carcinogenesis. Crit Rev Oncol Hemat 42:93–103. doi: 10.1016/S1040-8428(01)00212-8 CrossRefGoogle Scholar
  28. Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione-S-transferases—the first enzymatic step in mercapturic acid formation. J Biol Chem 249:7130–7139Google Scholar
  29. Kelly JM, Janz DM (2009) Assessment of oxidative stress and histopathology in juvenile northern pike (Esox lucius) inhabiting downstream of a uranium mill. Aquat Toxicol 92:240–249. doi: 10.1016/j.aquatox.2009.02.007 CrossRefGoogle Scholar
  30. Lalaouni A, Henderson C, Kupper C, Grant MH (2007) The interaction of chromium (VI) with macrophages: depletion of glutathione and inhibition of glutathione reductase. Toxicology 236(1–2):76–81. doi: 10.1016/j.tox.2007.04.002 CrossRefGoogle Scholar
  31. Lefcort H, Meguire RA, Wilson LH, Ettinger WF (1998) Heavy metals alter the survival, growth, metamorphosis, and antipredatory behaviour of Columbia spotted frog (Rana luteiventris) tadpoles. Arch Environ Contam Toxicol 35:447–456. doi: 10.1007/s002449900401 CrossRefGoogle Scholar
  32. Leonard SS, Harris GK, Shi X (2004) Metal-induced oxidative stress and signal transduction. Free Radical Bio Med 37(12):1921–1942. doi: 10.1016/j.freeradbiomed.2004.09.010 CrossRefGoogle Scholar
  33. Linder G, Grillitsch B (2000) Ecotoxicology of metals. In: Sparling DW, Linder G, Bishop CA (eds) Ecotoxicology of amphibians and reptiles. SETAC Technical Publication Series, Pensacola, FLGoogle Scholar
  34. Loumbourdis NS, Kyriakkopoulou-Skavounou P, Zachariadis G (1999) Effects of cadmium exposure on bioaccumulation and larval growth in the frog Rana ridibunda. Environ Pollut 104:429–433. doi: 10.1016/S0269-7491(98)00172-9 CrossRefGoogle Scholar
  35. Loumbourdis NS, Kostaropoulos I, Theodoropoulou B, Kalmanti D (2007) Heavy metal accumulation and metallothionein concentration in frog Rana ridibunda after exposure to chromium or a mixture of chromium and cadmium. Environ Pollut 145:787–792. doi: 10.1016/j.envpol.2006.05.011 CrossRefGoogle Scholar
  36. Lourenço JI, Pereira RO, Silva AC, Morgado JM, Carvalho FP, Oliveira JM, Malta MP, Paiva AA, Mendo SA, Gonçalves F (2011) Genotoxic endpoints in the earthworms sub-lethal assay to evaluate natural soils contaminated by metals and radionuclides. J Hazard Mater 186:786–795. doi: 10.1016/j.jhazmat.2010.11.073 CrossRefGoogle Scholar
  37. MA (1998) Decreto Lei no. 236/98, 1 Agosto. Ministério do Ambiente. Diário da Republica no. 176/98 Série I–A, pp 3676–3722.
  38. Malik N, Biswas AK, Qureshi A, Borana K, Virha R (2010) Bioaccumulation of heavy metals in fish tissues of freshwater Lake of Bhopal. Environ Monit Assess 160:267–276. doi: 10.1007/s10661-008-0693-8 CrossRefGoogle Scholar
  39. Marques SM, Gonçalves F, Pereira R (2008) Effects of a uranium mine effluent in the early-life stages of Rana perezi Seoane. Sci Total Environ 402:29–35. doi: 10.1016/j.scitotenv.2008.04.005 CrossRefGoogle Scholar
  40. Marques SM, Antunes SC, Pissarra H, Pereira ML, Gonçalves F, Pereira R (2009) Histopathological changes and erythrocytic nuclear abnormalities in Iberian green frogs (Rana perezi Seoane) from a uranium mine pond. Aquat Toxicol 91:187–195. doi: 10.1016/j.aquatox.2008.04.010 CrossRefGoogle Scholar
  41. Marqués MJ, Martínez-Conde E, Rovira JV (2003) Effects of zinc and lead mining on the benthic macroinvertebrates of a fluvial ecosystem. Water Air Soil Poll 148:363–388. doi: 10.1023/A:1025411932330 CrossRefGoogle Scholar
  42. McDiarmid RW, Mitchell JC (2000) Diversity and distribution of amphibians and reptiles. In: Sparling DW, Linder G, Bishop CA (eds) Ecotoxicology of amphibians and reptiles. SETAC Technical Publication Series, Pensacola, FLGoogle Scholar
  43. Meister A, Anderson ME (1983) Glutathione. Ann Rev Biochem 52:711–760CrossRefGoogle Scholar
  44. Oliveira JMS, Ávila PF (1998) Estudo geoquímico na área da mina da Cunha Baixa (Mangualde, no centro de Portugal). Relatório do Instituto Geológico e Mineiro, LisboaGoogle Scholar
  45. Oliveira JMS, Ávila PF (2001) Geoquímica na área envolvente da mina da Cunha Baixa (Mangualde, no centro de Portugal). Estudos, Notas e Trabalhos, Tomo 43, Instituto Geológico e MineiroGoogle Scholar
  46. Ortiz ME, Marco A, Saiz N, Lizana M (2004) Impact of ammonium nitrate on growth and survival of six european amphibians. Arch Environ Contam Toxicol 47:234–239. doi: 10.1007/s00244-004-2296-x CrossRefGoogle Scholar
  47. Pandya CD, Pillai PP, Gupta SS (2010) Lead and cadmium co-exposure mediated toxic insults on hepatic steroid metabolism and antioxidant system of adult male rats. Biol Trace Elem Res 134:307–317. doi: 10.1007/s12011-009-8479-6 CrossRefGoogle Scholar
  48. Pellmar TC, Fuciarelli AF, Ejnik JW, Hamilton M, Hogan J, Strocko S, Emond C, Mottaz HM, Landauer MR (1999) Distribution of uranium in rats implanted with depleted uranium pellets. Toxicol Sci 49:29–39. doi: 10.1093/toxsci/49.1.29 CrossRefGoogle Scholar
  49. Pereira R, Antunes SC, Marques SM, Gonçalves F (2008) Contribution for tier 1 of the ecological risk assessment of Cunha Baixa uranium mine (central Portugal): I. Soil chemical characterization. Sci Total Environ 390:377–386. doi: 10.1016/j.scitotenv.2007.08.051 CrossRefGoogle Scholar
  50. Pereira R, Marques CR, Silva Ferreira MJ, Neves MFJV, Caetano AL, Antunes SC, Mendo S, Gonçalves F (2009) Phytotoxicity and genotoxicity of soils from an abandoned uranium mine area. Appl Soil Ecol 42(3):209–220. doi: 10.1016/j.apsoil.2009.04.002 CrossRefGoogle Scholar
  51. Periyakaruppan A, Kumar F, Sarkar S, Sharma CS, Ramesh GT (2007) Uranium induces oxidative stress in lung epithelial cells. Arch Toxicol 81:389–395. doi: 10.1007/s00204-006-0167-0 CrossRefGoogle Scholar
  52. Pigeolet E, Corbisier P, Houbion A, Lambert D, Michiels C, Raes M, Zachary MD, Remacle J (1990) Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals. Mech Ageing Dev 51(3):283–297. doi: 10.1016/0047-6374(90)90078-T CrossRefGoogle Scholar
  53. Rossman MD (1994) Beryllium. In: Seiler HG, Sigel A, Sigel H (eds) Handbook on metals in clinical and analytical chemistry. Marcel Dekker, Inc, New YorkGoogle Scholar
  54. Różanowska M, Sarna T, Land EJ, Truscott TG (1999) Free radical scavenging properties of melanin interaction of eu- and pheo-melanin models with reducing and oxidising radicals. Free Radical Biol Med 26:518–525. doi: 10.1016/S0891-5849(98)00234-2 CrossRefGoogle Scholar
  55. Santo JC, Freire AP (1983) Tratamento de minérios pobres na mina da Cunha Baixa. Bol Minas 20(3):139–145Google Scholar
  56. Schaller K, Raithel H, Angerer J (1994a) Nickel. In: Seiler HG, Sigel A, Sigel H (eds) Handbook on metals in clinical and analytical chemistry. Marcel Dekker, Inc, New YorkGoogle Scholar
  57. Schaller K, Letzel S, Angerer J (1994b) Aluminum. In: Seiler HG, Sigel A, Sigel H (eds) Handbook on metals in clinical and analytical chemistry. Marcel Dekker, Inc, New YorkGoogle Scholar
  58. Singh MS, Sivalingam PM (1982) In vitro study on the interactive effects of heavy metals on catalase activity of Sarotherodon mossambicus (Peters). J Fish Biol 20(6):683–688. doi: 10.1111/j.1095-8649.1982.tb03978.x CrossRefGoogle Scholar
  59. Stohs SJ, Bagchi D (1995) Oxidative mechanisms in the toxicity of metal ions. Free Radical Biol Med 18(2):321–336. doi: 10.1016/0891-5849(94)00159-H CrossRefGoogle Scholar
  60. Stolyar OB, Loumbourdis NS, Falfushinska HI, Romanchuk LD (2008) Comparison of metal bioavailability in frogs from urban and rural sites of western Ukraine. Arch Environ Contam Toxicol 54:107–113. doi: 10.1007/s00244-007-9012-6 CrossRefGoogle Scholar
  61. Strydom C, Robinson C, Pretorius E, Whitcutt JM, Marx J, Bornman MS (2006) The effect of selected metals on the central metabolic pathways in biology: a review. Water SA 32(4):543–554Google Scholar
  62. Suter GW II, Tsao CL (1996) Toxicological benchmarks for screening potential contaminants of concern for effects on aquatic biota: revision. Oak Ridge National Laboratory, ES/ER/TM-96/R2. Oak Ridge National Laboratory, Oak Ridge, TNCrossRefGoogle Scholar
  63. Tandogan B, Ulusu NN (2010) Inhibition of purified bovine liver glutathione reductase with some metal ions. J Enzym Inhib Med Chem 25:68–73. doi: 10.3109/14756360903016512 CrossRefGoogle Scholar
  64. Valavanidis A, Vlahogianni T, Dassenakis M, Scoullos M (2006) Molecular biomarker of oxidative stress in aquatic organism in relation to toxic environmental pollutants. Ecotox Environ Safe 64(2):178–189. doi: 10.1016/j.ecoenv.2005.03.013 CrossRefGoogle Scholar
  65. Van der Oost R, Beyer J, Vermeulen NPE (2003) Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ Toxicol Pharmacol 13:57–149. doi: 10.1016/S1382-6689(02)00126-6 CrossRefGoogle Scholar
  66. Vassault A (1983) Lactate dehydrogenase. Method Enzym Anal 3:118–126Google Scholar
  67. Vogiatzis AK, Loumbourdis NS (1997) Uptake, tissue distribution and depuration of cadmium (Cd) in the frog Rana ridibunda. Bull Environ Contam Toxicol 59:770–776. doi: 10.1007/s001289900547 CrossRefGoogle Scholar
  68. Wolke RE (1992) Piscine macrophage aggregates: a review. Annu Rev Fish Dis 2:337–343. doi: 10.1016/0959-8030(92)90058-6 CrossRefGoogle Scholar
  69. Zar JH (1996) Biostatistical analysis, 3rd edn. Prentice-Hall International Inc, New JerseyGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Sérgio M. Marques
    • 1
    Email author
  • Sara C. Antunes
    • 1
  • Bruno Nunes
    • 1
    • 2
  • Fernando Gonçalves
    • 1
  • Ruth Pereira
    • 1
  1. 1.CESAM (Centro de Estudos do Ambiente e do Mar) & Departamento de Biologia, Universidade de AveiroAveiroPortugal
  2. 2.CIAGEB, FCS-UFP—Centro de Investigação em Alterações Globais, Energia e Bioengenharia, Faculdade de Ciências da Saúde da Universidade Fernando PessoaPortoPortugal

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