Ecotoxicology

, Volume 17, Issue 3, pp 153–163 | Cite as

Oxidative stress biomarkers and heart function in bullfrog tadpoles exposed to Roundup Original®

  • Monica J. Costa
  • Diana A. Monteiro
  • Abilio L. Oliveira-Neto
  • Francisco T. Rantin
  • Ana L. Kalinin
Article

Abstract

Oxidative stress biomarkers, in vivo heart rate (fH), and contraction dynamics of ventricle strips of bullfrog (Lithobates catesbeiana) tadpoles were evaluated after 48 h of exposure to a sub-lethal concentration (1 ppm) of the herbicide Roundup Original® (glyphosate 41%). The activities of the antioxidant enzymes superoxide dismutase and catalase were increased in the liver and decreased in muscle, while oxidative damage to lipids increased above control values in both tissues, showing that the generation of reactive oxygen species and oxidative stress are involved in the toxicity induced by Roundup®. Additionally, tadpoles’ hyperactivity was associated with tachycardia in vivo, probably due to a stress-induced adrenergic stimulation. Ventricle strips of Roundup®-exposed tadpoles (R-group) presented a faster relaxation and also a higher cardiac pumping capacity at the in vivo contraction frequency, indicating that bullfrog tadpoles were able to perform cardiac mechanistic adjustments to face Roundup®-exposure. However, the lower maximal in vitro contraction frequency of the R-group could limit its in vivo cardiac performance, when the adrenergic-stimulation is present. The association between the high energetic cost to counteract the harmful effects of this herbicide and the induction of oxidative stress suggest that low and realistic concentrations of Roundup® can have an impact on tadpoles’ performance and success, jeopardizing their survival and/or population establishment.

Keywords

Roundup® Oxidative stress biomarkers Cardiac contractility Bullfrog tadpoles Lithobates catesbeiana 

References

  1. Abdullah MP, Daud J, Hong KS, Yew CH (1995) Improved method for the determination of glyphosate in water. J Chromatogr A 697:363–369CrossRefGoogle Scholar
  2. Aebi H (1974) Catalase. In: Bergmayer HU (ed) Methods of enzymatic analysis. Academic Press, New York, pp 671–684Google Scholar
  3. Ahmad I, Pacheco M, Santos MA (2006) Anguilla anguilla L. oxidative stress biomarkers: an in situ study of freshwater wetland ecosystem (Pateira de Fermentelos, Portugal). Chemosphere 65:952–962CrossRefGoogle Scholar
  4. Ahmad I, Ahmada I, Hamidb T, Fatimaa M, Chandc HS, Jainb SK, Athara M, Raisuddina S (2000) Induction of hepatic antioxidants in freshwater catfish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure. Biochim Biophys Acta 1519:37–48Google Scholar
  5. American Veterinary Medical Association (2001) Report of the AVMA panel on euthanasia. J Am Vet Med Assoc 218:669–696CrossRefGoogle Scholar
  6. Andersen JB, Jensen FB, Wang T (2001) Effects of temperature and oxygen availability on circulating catecholamines in the toad Bufo marinus. Comp Biochem Physiol A 129:473–486CrossRefGoogle Scholar
  7. ASTM—American Society for Testing and Materials (2000) Standard guide for conducting acute toxicity tests on test materials with fishes, macroinvertebrates, and amphibians E 729–96. In: Annual Book of Standards, Vol 11.05, Section 11. Philadelphia, PA, pp 220–240Google Scholar
  8. Atli G, Alptekin Ö, Tükel S, Canli M (2006) Response of catalase activity to Ag+, Cd2+, Cr6+, Cu2+ and Zn2+ in five tissues of freshwater fish Oreochromis niloticus. Comp Biochem Physiol C 143:218–224CrossRefGoogle Scholar
  9. Avci A, Kaçmaz M, Durak İ (2005) Peroxidation in muscle and liver tissues from fish in a contaminated river due to a petroleum refinery industry. Ecotoxicol Environ Saf 6:101–105CrossRefGoogle Scholar
  10. Bagnyukovaa TV, Vasylkiva OY, Storeyb KB, Lushchaka VI (2005) Catalase inhibition by amino triazole induces oxidative stress in goldfish brain. Brain Res 1052:180–186CrossRefGoogle Scholar
  11. Banudevi S, Krishnamoorthy G, Venkatataman P, Vignesh C, Aruldhas MM, Arunakaran J (2006) Role of α-tocopherol on antioxidant status in liver, lung and kidney of PCB exposed male albino rats. Food Chem Toxicol 44:2040–2046CrossRefGoogle Scholar
  12. Bers DM (2001) Excitation-contraction coupling and cardiac contractile force, 2nd edn. Dordrecht, Kluwer Academic Publishers, p 258Google Scholar
  13. Boone MD, James SM (2003) Interactions of an insecticide, herbicide, and natural stressors in amphibian community mesocosms. Ecol Appl 13:829–841CrossRefGoogle Scholar
  14. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  15. Brausch JM, Smith PN (2007) Toxicity of three polyethoxylated tallowamine surfactant formulations to laboratory and field collected fairy shrimp, Thamnocephalus platyurus. Arch Environ Contam Toxicol 52(2):217–221CrossRefGoogle Scholar
  16. Bridges CM. (2000) Long-term effects of pesticide exposure at various life stages of the southern leopard frog (Rana sphenocephala). Arch Environ Contam Toxicol 39:91–96CrossRefGoogle Scholar
  17. Bridges CM (1997) Tadpole swimming performance and activity affected by acute exposure to sublethal levels of carbaryl. Env Toxicol Chem 16(9):1935–1939CrossRefGoogle Scholar
  18. Broomhall SD (2005) Measuring chemical impacts on amphibians: ecotoxicity and behavioural data in governmental regulation. Appl Herpetol 2:259–285CrossRefGoogle Scholar
  19. Burggren WW, Feder ME, Pinder A (1983) Temperature and the balance between aerial and aquatic respiration in larvae of Rana berlandieri and Rana catesbeiana. Physiol Zool 56:263–273Google Scholar
  20. Calore EE, Perez NM, Herman MM (2007) Morphometric studies of cardiac myocytes of rats chronically treated with an organophosphate. Ecotoxicol Environ Saf 66(3):447–450CrossRefGoogle Scholar
  21. Carvalho FT, Velini ED, Negrisoli E, Rossi CVS (2005) Eficácia do carfentrazone-ethyl no controle de plantas aquáticas latifoliadas em caixas-d’água. Planta Daninha 23(2):305–310Google Scholar
  22. Çavas T, Konen S (2007) Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay. Mutagenesis 22(4):263–268CrossRefGoogle Scholar
  23. Christin MS, Gendron AD, Brousseau P, Menard L, Marcogliese DJ, Cyr D, Ruby S, Fournier M (2003) Effects of agricultural pesticides on the immune system of Rana pipiens and on its resistance to parasitic infection. Environ Toxicol Chem 22:1127–1133CrossRefGoogle Scholar
  24. Clements C, Ralph S, Petras M (1997) Genotoxicity of select herbicides in Rana catesbeiana tadpoles using the alkaline single-cell gel DNA electrophoresis (Comet) assay. Environ Mol Mutagen 29(3):277–288CrossRefGoogle Scholar
  25. Collins JP, Storfer A (2003) Global amphibian declines: sorting the hypotheses. Divers Distrib 9:89–98CrossRefGoogle Scholar
  26. Comporti M (1985) Lipid peroxidation and cellular damage in toxic liver injury. Lab Investig 523:599–623Google Scholar
  27. Cox C, Surgan M (2006) Unidentified inert ingredients in pesticides: implications for human and environmental health. Environ Health Persp 114(12):1803–1806Google Scholar
  28. Crouch RK, Gandy SC, Kinsey G (1981) The inhibition of islet superoxide dismutase by diabetogenic drugs. Diabetes 30:235–241Google Scholar
  29. Di Giulio RT, Washiburn PC, Wenning RJ, Winston GW, Jewell CS (1989) Biochemical responses in aquatic animals: a review of determinants of oxidative stress. Environ Toxocol Chem 8:1103–1123CrossRefGoogle Scholar
  30. Dorval J, Hontela A (2003) Role of glutathione redox cycle and catalase in defense against oxidative stress induced by endosulfan in adrenocortical cells of rainbow trout (Oncorhynchus mykiss). Toxicol Appl Pharmacol 192:191–200CrossRefGoogle Scholar
  31. Driedzic WR, Gesser H (1994) Energy metabolism and contractility in ectothermic vertebrate hearts: hypoxia, acidosis, and low temperature. Physiol Rev 74(1): 221–258Google Scholar
  32. Erlij D, Cetrangolo R, Valadez R (1965) Adrenotropic receptors in the frog. J Pharmacol Exp Ther 149:65–70Google Scholar
  33. Farrell AP, Jones DR (1992) The Heart. In: Hoar WS, Randall DJ, Farrell AP (eds) Fish physiology: the cardiovascular system v 12A. Academic Press Inc., New York, pp 1–88Google Scholar
  34. Favari L, Lopez E, Martinez-Tabche L, Diaz-Pardo E (2002) Effect of insecticides on plankton and fish of Ignacio Ramirez reservoir (Mexico): a biochemical and biomagnification study. Ecotoxicol Environ Saf 51:177–186CrossRefGoogle Scholar
  35. Feder ME (1983) Responses to acute aquatic hypoxia in larvae of the frog Rana beriandieri. J Exp Biol 104:79–95Google Scholar
  36. Fulle S, Protasi F, Di Tano G, Pietrangelo T, Beltramin A, Boncompagni S, Vecchiet L, Fano G (2004) The contribution of reactive oxygen species to sarcopenia and muscle ageing. Exp Gerontol 39:17–24CrossRefGoogle Scholar
  37. Gehin A, Guyon C, Nicod L (2006) Glyphosate-induced antioxidant imbalance in HaCaT: the protective effect of vitamins C and E. Environ Toxicol Pharmacol 22:27–34CrossRefGoogle Scholar
  38. Georgieva NV (2005) Oxidative stress as a factor of disrupted ecological oxidative balance in biological systems – a review. Bulg J Vet Med 8(1):1–11Google Scholar
  39. Giesy JP, Dobson S, Solomon KR (2000) Ecotoxicological risk assessment for Roundup herbicide. Rev Contam Toxicol 167:35–120Google Scholar
  40. González A, Marín O, Tuinhof R, Smeets WJAJ (2004) Ontogeny of catecholamine systems in the central nervous system of anuran amphibians: an immunohistochemical study with antibodies against tyrosine hydroxylase and dopamine. J Comp Neurol 346(1):63–79CrossRefGoogle Scholar
  41. Gül S, Belge-Kurutas E, Yildiz E, Sahan A, Doran F (2004) Pollution correlated modifications of liver antioxidant systems and histopathology of fish (Cyprinidae) living in Seyhan Dan Lake. Turkey Environ Int 30:605–609CrossRefGoogle Scholar
  42. Gurushankara HP, Krishnamurthy SV, Vasudev V (2007) Effect of malathion on survival, growth, and food consumption of Indian cricket frog (Limnonectus limnocharis) tadpoles. Arch Environ Contam Toxicol 52:251–256CrossRefGoogle Scholar
  43. Halliwell B, Gutteridge JMC (1987) Oxygen toxicity, oxygen radicals, transition metals and disease. J Biochem 219:1–14Google Scholar
  44. Halliwell B, Gutteridge JMC (1989) Free radicals in biology and medicine, 2nd edn. Claredon Press, Oxford, p 469Google Scholar
  45. Hazarika A, Sarkar SN, Hajare S, Kataria M, Malik JK (2003) Influence of malathion pretreatment on the toxicity of anilofos in male rats: a biochemical interaction study. Toxicology 185:1–8CrossRefGoogle Scholar
  46. Hedrick MS, Chen AK, Jessop KL (2005) Nitric oxide changes its role as a modulator of respiratory motor activity during development in the bullfrog (Rana catesbeiana). Comp Biochem Physiol A 142:231–240CrossRefGoogle Scholar
  47. Herman CA, Sandoval EJ (1983) Catecholamine effects on blood pressure and heart rate in the American bullfrog, Rana catesbeiana. Gen Comp Endocrinol 52:142–148CrossRefGoogle Scholar
  48. Hillman SS (1976) Cardiovascular correlates of maximal oxygen consumption rates in anuran amphibians. J Com Physiol 109:199–207Google Scholar
  49. Hillman SS, Withers PC, Hedrick MS, Kimmel PB (1985) The effects of erythrocythemia on blood viscosity, maximal systemic oxygen transport capacity and maximal rates of oxygen consumption in an amphibian. J Com Physiol 155:577–581Google Scholar
  50. Howe CM, Berrill M, Pauli BD, Helbing CC, Werry K, Veldhoen N (2004) Toxicity of glyphosate-based pesticides to four. North American Frog Species Environ Toxicol Chem 23(8):1928–1938Google Scholar
  51. Hultberg M (2007) Cysteine turnover in human cell liones is influenced by glyphosate. Environ Toxicol Pharmacol 23:19–22CrossRefGoogle Scholar
  52. Jia XX, Burggren WW (1997) Developmental changes in chemoreceptive control of gill ventilation in larval bullfrogs (Rana catesbeiana). II. Sites of O2-sensitive chemoreceptors. J Exp Biol 200:2237–2248Google Scholar
  53. Jiang ZY, Woollard ACS, Wolff SP (1991) Lipid hydroperoxide measurement by oxidation of Fe2+ in the presence of xylenol orange: comparison with the TBA assay and iodometric method. Lipids 26:777–860CrossRefGoogle Scholar
  54. Jiang ZY, Hunt JV, Wolff SP (1992) Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low-density lipoprotein. Anal Biochem 202(2):384–389CrossRefGoogle Scholar
  55. Jimenez BD, Stegeman JJ (1990) Detoxication enzymes as indicators of environmental stress on fish. In: Adams SM (ed) Biological indicators of stress in fish, vol 8. American Fisheries Symposium. Bethesda, Maryland, pp 67–79Google Scholar
  56. Johansson M, Piha H, Kylin H, Merila J (2006) Toxicity of six pesticides to common frog (Rana temporaria) tadpoles. Environ Toxicol Chem 25(12):3164–3170CrossRefGoogle Scholar
  57. Kavitha P, Rao V (2007) Oxidative stress and locomotor behaviour response as biomarkers for assessing recovery status of mosquito Wsh, Gambusia aYnis after lethal eVect of an organophosphate pesticide, monocrotophos. Pest Biochem Physiol 87:182–188CrossRefGoogle Scholar
  58. Kehrer JP (1993) Free radicals as mediators of tissue injury and disease. Crit Rev Toxicol 34:21–48CrossRefGoogle Scholar
  59. Kimmel KB (2004) Adrenergic receptors and the regulation of vascular resistance in bullfrog tadpoles (Rana catesbeiana). J Comp Physiol B 162(5):455–462Google Scholar
  60. Kirby S, Burnstock G (1969) Pharmacological studies of the cardiovascular system in the anaesthetized sleepy lizard (Tiliqua rugosa) and toad (Bufo marinus). Comp Biochem Physiol 28:321–331CrossRefGoogle Scholar
  61. Kloberg AJ, Fritsche R (2002) Catecholamines are present in larval Xenopus laevis: a potential source for cardiac control. J Exp Zool 292(3):293–303CrossRefGoogle Scholar
  62. Koyama Ka., Koyama Ky, Goto K (1997) Cardiovascular effects of a herbicide containing glufosinate and a surfactant: in vitro and in vivo analyses in rats. Toxicol Appl Pharmacol 145:409–414CrossRefGoogle Scholar
  63. Kruger NJ (1994) The Bradford method for protein quantification. Meth Mol Biol 32:9–15Google Scholar
  64. Lambert MRK (1997) Effect of pesticides on amphibians and reptiles in sub-Saharan Africa. Rev Environ Contam Toxicol 150:31–73Google Scholar
  65. Layland J, Li JM, Shah AM (2002) Role of cyclic GMP-dependent protein kinase in the contractile response to exogenous nitric oxide in rat cardiac myocytes. J Physiol 540:457–467CrossRefGoogle Scholar
  66. Leonard AWR, Hyne V, Lim RP, Chapman JC (1999) Effect of endosulfan runoff from cotton fields on macroinvertebrates in the Namoi River. Ecotoxicol Environ Saf 42:125–134CrossRefGoogle Scholar
  67. Lewatowski B, Pytkowski B (1987) Cellular mechanisms of the relationship between myocardial force and frequency of contractions. Prog Biophys Mol Biol 50:97–120CrossRefGoogle Scholar
  68. Lillo RS (1979) Autonomic cardiovascular control during submergence and emergence in bullfrogs. Am J Physiol 237:R210–R216Google Scholar
  69. Liu Y, Zhang Y, Liu J, Huang D (2006) The role of reactive oxygen species in the herbicide acetochlor-induced DNA damage on Bufo raddei tadpole liver. Aquatic Toxicol 78:21–26CrossRefGoogle Scholar
  70. Matikainen N, Vornanen M (1992) Effect of season and temperature acclimation on the function of crucian carp (Carassius carassius) heart. J Exp Biol 167:203–220Google Scholar
  71. Monsanto Company (1991) Roundup herbicide, material safety data sheet. Monsanto Canada Streetsville, ON, CanadaGoogle Scholar
  72. Monteiro DA, Almeida JA, Rantin FT, Kalinin AL (2006) Oxidative stress biomarkers in the freshwater characid fish, Brycon cephalus, exposed to organophosphorus insecticide Folisuper 600 (methyl parathion). Comp Biochem Physiol 143:141–149CrossRefGoogle Scholar
  73. Oruç EO, Usta D (2007) Evaluation of oxidative stress responses and neurotoxicity potential of diazinon in different tissues of Cyprinus carpio. Environ Toxicol Pharmacol 23:48–55CrossRefGoogle Scholar
  74. Otero J, Toni P, Garcia-Morato YV (1983) Superoxo dismutase: método para su determinación. Rev Iberoam Invest Clin 2:121–127Google Scholar
  75. Peña-Llopis S, Ferrando MD, Peña JB (2003) Fish tolerance to organophosphate-induced oxidative stress is dependent on the glutathione metabolism and enhanced by N-acetylcysteine. Aquat Toxicol 65:337–360Google Scholar
  76. Perkins PJ, Herman J, Boermans HJ, Stephenson GR (2000) Toxicity of glyphosate and triclopyr using the frog embryo teratogenesis assay-Xenopus. Environ Toxicol Chem 19(4):940–945CrossRefGoogle Scholar
  77. Pieniazek D, Bukowska B, Duda W (2004) Comparison of the effect of Roundup Ultra 360 SL pesticide and its active compound glyphosate on human erythrocytes. Pest Biochem Physiol 79:58–63CrossRefGoogle Scholar
  78. Relyea RA (2003) Predator cues and pesticides: A double dose of danger for amphibians. Ecol Appl 13(6):1515–1521CrossRefGoogle Scholar
  79. Relyea RA (2004) Growth and survival of five amphibian species exposed to combinations of pesticides. Environ Toxicol Chem 23(7):1737–1742CrossRefGoogle Scholar
  80. Relyea RA (2005a) The lethal impact of Roundup on aquatic and terrestrial amphibians. Ecol Appl 15:1118–112CrossRefGoogle Scholar
  81. Relyea RA (2005b) The impact of insecticides and herbicides on the biodiversity and productivity of aquatic communities. Ecol Appl 15(2):618–627CrossRefGoogle Scholar
  82. Relyea RA (2006) Letters to the editor: the impact of insecticides and herbicides on the biodiversity and productivity of aquatic communities: response. Ecol Appl 16(5):2027–2034CrossRefGoogle Scholar
  83. Risso-de-Faverney C, Devaux A, Lafaurie M, Girard JP, Bailly B, Rahmani R (2001) Cadmium induces apoptosis and genotoxicity in rainbow trout hepatocytes through generation of reactive oxygen species. Aquat Toxicol 53:65–76CrossRefGoogle Scholar
  84. Rodrigues BN, Almeida FLS (2005) Guia de Herbicidas, 5th edn. Londrina, Grafmark, p 592Google Scholar
  85. Saygili EI, Konukoglu D, Papila S, Aksay T (2003) Levels of plasma vitamin E, vitamin C, TBARS and cholesterol in male patients with colorectal tumors. Biochemistry (Moscow) 68(3):325–328CrossRefGoogle Scholar
  86. Schwerte T, Printz E, Regina Fritsche R (2002) Vascular control in larval Xenopus laevis: the role of endothelial-derived factors. J Exp Biol 205:225–232Google Scholar
  87. Shiels H, Farrell AP (1997) The effect of temperature and adrenaline on the relative importance of the sarcoplasmic reticulum in contributing Ca2+ to force development in isolated ventricular trabeculae from rainbow trout. J Exp Biol 1(200):1607–1621Google Scholar
  88. Sk UH, Bhattacharya S (2006) Prevention of cadmium induced lipid peroxidation, depletion of some antioxidative enzymes and glutathione by series of novel organoselenocyanates Environ. Toxicol Pharmacol 22:298–308Google Scholar
  89. Smith GR (2001) Effects of acute exposure to a commercial formulation of glyphosate on the tadpoles of two species of anurans. Bull Environ Contam Toxicol 67:483–488CrossRefGoogle Scholar
  90. Sparling DW, Matson C, Bickham J, Doelling-Brown P (2006) Toxicity of glyphosate as Glypro and LI700 to red-eared slider (Trachemys scripta elegans) embryos and early hatchlings. Environ Toxicol Chem 25(10):2768–2774CrossRefGoogle Scholar
  91. Sys SU, Pellegrino D, Mazza R, Gattuso A, Andries LJ, Tota B (1997) Endocardial endothelium in the avascular heart of the frog: Morphology and role of nitric oxide. J Exp Biol 200:3109–3118Google Scholar
  92. Szarek J, Siwicki A, Andrzejewska A, Terech-Majewska E, Banaszkiewicz T (2000) Effects of the herbicide Roundup on the ultrastructural pattern of hepatocytes in carp (Cyprinus carpio). Mar Environ Res 50(1–5):263–266CrossRefGoogle Scholar
  93. Terech-Majewska E, Siwicki AK, Szweda W (2004) Modulative influence of lysozyme dimer on defense mechanisms in the carp (Cyprinus carpio) and European sheatfish (Silurus glanis) after suppression induced by herbicide. Roundup Pol J Vet Sci 7(2):123–128Google Scholar
  94. Thompson DG, Wojtaszek BF, Staznik B, Chartrand DT, Stephenson GR (2004) Chemical and biomonitoring to assess potential acute effects of Vision herbicide on native amphibian larvae in forest wetlands. Environ Toxicol Chem 23:843–849CrossRefGoogle Scholar
  95. Thompson DG, Solomon KR, Wojtaszek BF, Edginton AN, Stephenson GR (2006) Letters to the editor: the impact of insecticides and herbicides on the biodiversity and productivity of aquatic communities. Ecol Appl 16(5):2022–2027CrossRefGoogle Scholar
  96. Tsui MTK, Chu LM (2003) Aquatic toxicity of glyphosate-based formulations: comparison between different organisms and the effects of environmental factors. Chemosphere 52(7):1189–1197CrossRefGoogle Scholar
  97. Venturino A, Rosenbaum E, De Castro AC, Anguiano OL, Gauna L, De Schroeder TF, D’Angelo AMP (2003) Biomarkers of effect in toads and frogs. Biomarkers 8:167–186CrossRefGoogle Scholar
  98. Wassersug RJ, Paul RD, Feder ME (1981) Cardiorespiratory synchrony in anuran larvae (Xenopus laevis, Pachymedusa dacnicolor, Rana berlandieri). Comp Biochem Physiol 70A:329–334CrossRefGoogle Scholar
  99. Withers PC, Hillman SS (2001) Allometric and ecological relationships of ventricle and liver mass in anuran amphibians. Functional Ecol 15:60–69CrossRefGoogle Scholar
  100. Wilhelm-Filho D, Giulivi C, Boveris A (1993) Antioxidant defenses in marine fish – I. Teleosts Comp Biochem Physiol C 160:409–413Google Scholar
  101. Winston GW (1991) Oxidants and antioxidants in aquatic animals. Comp Biochem Physiol 100C:173–176Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Monica J. Costa
    • 1
  • Diana A. Monteiro
    • 2
  • Abilio L. Oliveira-Neto
    • 3
  • Francisco T. Rantin
    • 2
  • Ana L. Kalinin
    • 2
  1. 1.Campus of SorocabaFederal University of São CarlosSorocabaBrazil
  2. 2.Department of Physiological SciencesFederal University of São CarlosSao CarlosBrazil
  3. 3.Center of High Technological EducationCampinas State UniversityLimeiraBrazil

Personalised recommendations