Advertisement

Neurotoxicity Research

, Volume 19, Issue 3, pp 472–483 | Cite as

Single-Cell ELISA and Flow Cytometry as Methods for Highlighting Potential Neuronal and Astrocytic Toxicant Specificity

  • E. K. WoehrlingEmail author
  • E. J. Hill
  • E. E. Torr
  • M. D. Coleman
Article

Abstract

The timeline imposed by recent worldwide chemical legislation is not amenable to conventional in vivo toxicity testing, requiring the development of rapid, economical in vitro screening strategies which have acceptable predictive capacities. When acquiring regulatory neurotoxicity data, distinction on whether a toxic agent affects neurons and/or astrocytes is essential. This study evaluated neurofilament (NF) and glial fibrillary acidic protein (GFAP) directed single-cell (S-C) ELISA and flow cytometry as methods for distinguishing cell-specific cytoskeletal responses, using the established human NT2 neuronal/astrocytic (NT2.N/A) co-culture model and a range of neurotoxic (acrylamide, atropine, caffeine, chloroquine, nicotine) and non-neurotoxic (chloramphenicol, rifampicin, verapamil) test chemicals. NF and GFAP directed flow cytometry was able to identify several of the test chemicals as being specifically neurotoxic (chloroquine, nicotine) or astrocytoxic (atropine, chloramphenicol) via quantification of cell death in the NT2.N/A model at cytotoxic concentrations using the resazurin cytotoxicity assay. Those neurotoxicants with low associated cytotoxicity are the most significant in terms of potential hazard to the human nervous system. The NF and GFAP directed S-C ELISA data predominantly demonstrated the known neurotoxicants only to affect the neuronal and/or astrocytic cytoskeleton in the NT2.N/A cell model at concentrations below those affecting cell viability. This report concluded that NF and GFAP directed S-C ELISA and flow cytometric methods may prove to be valuable additions to an in vitro screening strategy for differentiating cytotoxicity from specific neuronal and/or astrocytic toxicity. Further work using the NT2.N/A model and a broader array of toxicants is appropriate in order to confirm the applicability of these methods.

Keywords

GFAP Neurofilaments NT2 Astrocytes Neurons Neurotoxicity 

Notes

Acknowledgments

The authors are grateful to the Humane Research Trust (Stockport, UK) for financial support for this study.

References

  1. Abreu-Villaca Y, Seidler FJ, Qiao D, Slotkin TA (2005) Modeling the developmental neurotoxicity of nicotine in vitro: cell acquisition, growth and viability in PC12 cells. Brain Res Dev Brain Res 154:239–246CrossRefPubMedGoogle Scholar
  2. AM ECV (2002) Target organ and target system toxicity. Altern Lab Anim 30(1):71–82Google Scholar
  3. Anderl JL, Redpath S, Ball AJ (2009) A neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity. J Vis Exp. doi: 10.3791/1173
  4. Andrews PW (1984) Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell-line in vitro. Dev Biol 103:285–293CrossRefPubMedGoogle Scholar
  5. Anthony DC, Montine TJ, Valentine WM, Graham DG (2001) Chapter 16: toxic responses of the nervous system. In: Klassen CD (ed) Casarett and Doull’s Toxicology: the basic science of poisons, 6th edn. McGraw–Hill, London, pp 535–564Google Scholar
  6. Araque A, Martin ED, Perea G, Arellano JI, Buno W (2002) Synaptically released acetylcholine evokes Ca2+ elevations in astrocytes in hippocampal slices. J Neurosci 22:2443–2450PubMedGoogle Scholar
  7. Atterwill CK, Bruinink A, Drejer J, Duarte E, Abdulla EM, Meredith C et al (1994) In-vitro neurotoxicity testing—the report and recommendations of ECVAM workshop-3. ATLA Altern Lab Anim 22:350–362Google Scholar
  8. Bal-Price AK, Suñol C, Weiss DG et al (2008) Application of in vitro neurotoxicity testing for regulatory purposes: Symposium III summary and research needs. Neurotoxicology 29:520–531CrossRefPubMedGoogle Scholar
  9. Bal-Price AK, Hogberg HT, Buzanska L, Coecke S (2009) Relevance of in vitro neurotoxicity testing for regulatory requirements: challenges to be considered. Neurotoxicol Teratol. doi: 10.1016/j.ntt.2008.12.003
  10. Bani-Yaghoub M, Felker JM, Naus CCG (1999) Human NT2/D1 cells differentiate into functional astrocytes. Neuroreport 10:3843–3846CrossRefPubMedGoogle Scholar
  11. Barbon A, Vallini I, La Via L, Marchina E (2003) Barlati S Glutamate receptor RNA editing: a molecular analysis of GluR2, GluR5 and GluR6 in human brain tissues and in NT2 cells following in vitro neural differentiation. Mol. Brain Res 117:168–178CrossRefPubMedGoogle Scholar
  12. Bechtel LK, Haverstick DM, Holstege CP (2008) Verapamil toxicity dysregulates the phosphatidylinositol 3-kinase pathway. Acad Emerg Med 15:368–374CrossRefPubMedGoogle Scholar
  13. Black AM, Pandya S, Clark D, Armstrong EA, Yager JY (2008) Effect of caffeine and morphine on the developing pre-mature brain. Brain Res 1219:136–142CrossRefPubMedGoogle Scholar
  14. Bruinink A, Zimmermann G, Riesen F (1991) Neurotoxic effects of chloroquine invitro. Arch Toxicol 65:480–484CrossRefPubMedGoogle Scholar
  15. Bunnemann B, Terron A, Zantedeschi V, Pich EM, Chiamulera C (2000) Chronic nicotine treatment decreases neurofilament immunoreactivity in the rat ventral tegmental area. Eur J Pharmacol 393:249–253CrossRefPubMedGoogle Scholar
  16. Carrier RL, Ma TC, Obrietan K, Hoyt KR (2006) A sensitive and selective assay of neuronal degeneration in cell culture. J Neurosci Meth 154:239–244CrossRefGoogle Scholar
  17. Cauli O, Morelli M (2005) Caffeine and the dopaminergic system. Behav Pharmacol 16:63–77CrossRefPubMedGoogle Scholar
  18. Chen JH, Wu KY, Chiu IM, Tsou TC, Chou CC (2009) Acrylamide-induced astrogliotic and apoptotic responses in human astrocytoma cells. Toxicol In Vitro 23:855–861CrossRefPubMedGoogle Scholar
  19. Chung RS, McCormack GH, King AE, West AK, Vickers JC (2005) Glutamate induces rapid loss of axonal neurofilament proteins from cortical neurons in vitro. Exp Neurol 193:481–488CrossRefPubMedGoogle Scholar
  20. Coecke S, Eskes C, Gartlon J, Kinsner A, Price A, van Vliet E et al (2006) The value of alternative testing for neurotoxicity in the context of regulatory needs. Environ Toxicol Pharmacol 21:153–167CrossRefGoogle Scholar
  21. Cookson MR, Pentreath VW (1994) Alterations in the glial fibrillary acidic protein-content of primary astrocyte cultures for evaluation of glial-cell toxicity. Toxicol In Vitro 8:351–356CrossRefPubMedGoogle Scholar
  22. Cookson MR, Thatcher NM, Ince PG, Shaw PJ (1996) Selective loss of neurofilament proteins after exposure of differentiated human IMR-32 neuroblastoma cells to oxidative stress. Brain Res 738:162–166CrossRefPubMedGoogle Scholar
  23. DeJongh J, Nordin-Andersson M, Ploeger BA, Forsby A (1999) Estimation of systemic toxicity of acrylamide by integration of in vitro toxicity data with kinetic simulations. Toxicol Appl Pharmacol 158:261–268CrossRefPubMedGoogle Scholar
  24. Desagher S, Glowinski J, Premont J (1996) Astrocytes protect neurons from hydrogen peroxide toxicity. J Neurosci 16:2553–2562PubMedGoogle Scholar
  25. El-Fawal HAN, O’Callaghan JP (2008) Autoantibodies to neurotypic and gliotypic proteins as biomarkers of neurotoxicity: assessment of trimethyltin (TMT). Neurotoxicology 29:109–115CrossRefPubMedGoogle Scholar
  26. Eng LF, Ghirnikar RS, Lee YL (2000) Glial fibrillary acidic protein: GFAP-thirty-one years (1969–2000). Neurochem Res 25:1439–1451CrossRefPubMedGoogle Scholar
  27. Exon JH (2006) A review of the toxicology of acrylamide. J Toxicol Environ Health B Crit Rev 9:397–412CrossRefPubMedGoogle Scholar
  28. Forsby A, Blaauboer B (2007) Integration of in vitro neurotoxicity data with biokinetic modelling for the estimation of in vivo neurotoxicity. Hum Exp Toxicol 26:333–338CrossRefPubMedGoogle Scholar
  29. Forsby A, Bal-Price AK, Camins A, Coecke S, Fabre N, Gustafsson H et al (2009) Neuronal in vitro models for the estimation of acute systemic toxicity. Toxicol In Vitro 23:1564–1569CrossRefPubMedGoogle Scholar
  30. Fredholm BB (2007) Adenosine, an endogenous distress signal, modulates tissue damage and repair. Cell Death Differ 14:1315–1323CrossRefPubMedGoogle Scholar
  31. Gartlon J, Kinsner A, Bal-Price A, Coecke S, Clothier RH (2006) Evaluation of a proposed in vitro test strategy using neuronal and non-neuronal cell systems for detecting neurotoxicity. Toxicol In Vitro 20:1569–1581CrossRefPubMedGoogle Scholar
  32. Gegg ME, Beltran B, Salas-Pino S, Bolanos JP, Clark JB, Moncada S et al (2003) Differential effect of nitric oxide on glutathione metabolism and mitochondrial function in astrocytes and neurons: implications for neuroprotection/neurodegeneration? J Neurochem 86:228–237CrossRefPubMedGoogle Scholar
  33. Gelinas S, Chapados C, Beauregard M, Gosselin I, Martinoli MG (2000) Effect of oxidative stress on stability and structure of neurofilament proteins. Biochem Cell Biol 78:667–674CrossRefPubMedGoogle Scholar
  34. Guan ZZ, Yu WF, Nordberg A (2003) Dual effects of nicotine on oxidative stress and neuroprotection in PC12 cells. Neurochem Int 43:243–249CrossRefPubMedGoogle Scholar
  35. Guerri C, Pascual M, Renau-Piqueras J (2001) Glia and fetal alcohol syndrome. Neurotoxicology 22:593–599CrossRefPubMedGoogle Scholar
  36. Guizzetti M, Moller T, Costa LG (2003) Ethanol inhibits muscarinic receptor-mediated DNA synthesis and signal transduction in human fetal astrocytes. Neurosci Lett 344:68–70CrossRefPubMedGoogle Scholar
  37. Haorah J, Ramirez SH, Floreani N, Gorantla S, Morsey B, Persidsky Y (2008) Mechanism of alcohol-induced oxidative stress and neuronal injury. Free Radic Biol Med 45:1542–1550CrossRefPubMedGoogle Scholar
  38. Harry GJ, Billingsley M, Bruinink A, Campbell IL, Classen W, Dorman DC et al (1998) In vitro techniques for the assessment of neurotoxicity. Environ Health Perspect 106:131–158CrossRefPubMedGoogle Scholar
  39. Herschman ZJ, Silverstein J, Blumberg G, Lehrfield A (1991) Central-nervous-system toxicity from nebulized atropine sulfate. J Toxicol Clin Toxicol 29:273–277CrossRefPubMedGoogle Scholar
  40. Holden LJ, Coleman MD (2008) Further preliminary assessment of three human glioma cell lines as models of human astrocytic toxicity in vitro. Env Toxicol Pharmacol 26:290–296CrossRefGoogle Scholar
  41. Kang SH, Lee YA, Won SJ, Rhee KH, Gwag BJ (2002) Caffeine-induced neuronal death in neonatal rat brain and cortical cell cultures. Neuroreport 13:1945–1950CrossRefPubMedGoogle Scholar
  42. Kinsner-Ovaskainen A, Rzepka R, Rudowski R, Coecke S, Cole T, Prieto P (2009) Acutoxbase, an innovative database for in vitro acute toxicity studies. Toxicol In Vitro 23:476–485CrossRefPubMedGoogle Scholar
  43. Kirkpatrick L, Brady S (1998) The cytoskeleton of neurons and glia, chap 11. In: Siegel GJ, Agranoff BW, Albers RW, Fisher SK, Uhler MD (eds) Basic neurochemistry: molecular, cellular and medical aspects, 6th edn. Lippincott, Williams and Wilkins, London, pp 135–174Google Scholar
  44. Knight A (2008) Non-animal methodologies within biomedical research and toxicity testing. Altex Altern Tierexp 25:213–231Google Scholar
  45. Kong CT, Holt DE, Ma SK, Lie AKW, Chan LC (2000) Effects of antioxidants and a caspase inhibitor on chloramphenicol-induced toxicity of human bone marrow and HL-60 cells. Hum Exp Toxicol 19:503–510CrossRefPubMedGoogle Scholar
  46. Kostrzewa RM, Segura-Aguilar J (2003) Novel mechanisms and approaches in the study of neurodegeneration and neuroprotection. A review. Neurotox Res 5:375–383CrossRefPubMedGoogle Scholar
  47. Lilienblum W, Dekant W, Foth H, Gebel T, Hengstler JG, Kahl R et al (2008) Alternative methods to safety studies in experimental animals: role in the risk assessment of chemicals under the new European Chemicals Legislation (REACH). Arch Toxicol 82:211–236CrossRefPubMedGoogle Scholar
  48. LoPachin RM, Aschner M (1993) Glial neuronal interactions—relevance to neurotoxic mechanisms. Toxicol Appl Pharmacol 118:141–158CrossRefPubMedGoogle Scholar
  49. LoPachin RM, Gavin T (2008) Acrylamide-induced nerve terminal damage: Relevance to neurotoxic and neurodegenerative mechanisms. J Agric Food Chem 56:5994–6003CrossRefPubMedGoogle Scholar
  50. LoPachin RM, Schwarcz AI, Gaughan CL, Mansukhani S, Das S (2004) In vivo and in vitro effects of acrylamide on synaptosomal neurotransmitter uptake and release. Neurotoxicology 25:349–363CrossRefPubMedGoogle Scholar
  51. Monnet-Tschudi F, Zurich MG, Honegger P (2007) Neurotoxicant-induced inflammatory response in three-dimensional brain cell cultures. Hum Exp Toxicol 26:339–346CrossRefPubMedGoogle Scholar
  52. Moore DJ, Chambers JK, Murdock PR, Emson PC (2002) Human Ntera-2/D1 neuronal progenitor cells endogenously express a functional P2Y1 receptor. Neuropharmacology 43:966–978CrossRefPubMedGoogle Scholar
  53. Mottin S, Laporte P, Cespuglio R (2003) Inhibition of NADH oxidation by chloramphenicol in the freely moving rat measured by picosecond time-resolved emission spectroscopy. J Neurochem 84:633–642CrossRefPubMedGoogle Scholar
  54. Newman MB, Kuo YP, Lukas RJ, Sanberg PR, Shytle RD, McGrogan MP, Zigova T (2002) Nicotinic acetylcholine receptors on NT2 precursor cells and hNT (NT2-N) neurons. Dev Brain Res 139:73–86CrossRefGoogle Scholar
  55. O’Callaghan JP, Sriram K (2005) Glial fibrillary acidic protein and related glial proteins as biomarkers of neurotoxicity. Expert Opin Drug Saf 4:433–442CrossRefPubMedGoogle Scholar
  56. O’Callaghan JP, Jensen KF, Miller DB (1995) Quantitative aspects of drug and toxicant-induced astrogliosis. Neurochem Int 26:115–124CrossRefPubMedGoogle Scholar
  57. O’Shaughnessy TJ, Zim B, Ma W, Shaffer KM, Stenger DA, Zamani K et al (2003) Acute neuropharmacologic action of chloroquine on cortical neurons in vitro. Brain Res 959:280–286CrossRefPubMedGoogle Scholar
  58. OECD Guideline for the testing of chemicals (1995) TG 418. Delayed neurotoxicity of organophosphorus substances following acute exposure, July 1995Google Scholar
  59. OECD Guideline for the testing of chemicals (1997) TG 424. Neurotoxicity study in rodents, July 1997Google Scholar
  60. OECD Guideline for the testing of chemicals (2007) TG 426. Developmental neurotoxicity study, October 2007Google Scholar
  61. Park J, Kwon D, Choi C, Oh JW, Benveniste EN (2003) Chloroquine induces activation of nuclear factor-kappa B and subsequent expression of pro-inflammatory cytokines by human astroglial cells. J Neurochem 84:1266–1274CrossRefPubMedGoogle Scholar
  62. Park JS, Choi KS, Jeong EJ, Kwon DH, Benveniste EN, Choi CH (2004) Reactive oxygen species mediate chloroquine-induced expression of chemokines by human astroglial cells. Glia 47:9–20CrossRefPubMedGoogle Scholar
  63. Park BC, Park SH, Paek SH, Park SY, Kwak MK, Choi HG et al (2008) Chloroquine-induced nitric oxide increase and cell death is dependent on cellular GSH depletion in A172 human glioblastoma cells. Toxicol Lett 178:52–60CrossRefPubMedGoogle Scholar
  64. Qiao D, Seidler FJ, Slotkin TA (2005) Oxidative mechanisms contributing to the developmental neurotoxicity of nicotine and chlorpyrifos. Toxicol Appl Pharmacol 206:17–26CrossRefPubMedGoogle Scholar
  65. Robenshtok E, Luria S, Tashma Z, Hourvitz A (2002) Adverse reaction to atropine and the treatment of organophosphate intoxication. Isr Med Assoc J 4:535–539PubMedGoogle Scholar
  66. Sandhu JK, Pandey S, Ribecco-Lutkiewicz M, Monette R, Borowy-Borowski H, Walker PR et al (2003) Molecular mechanisms of glutamate neurotoxicity in mixed cultures of NT2-derived neurons and astrocytes: Protective effects of coenzyme Q(10). J Neurosci Res 72:691–703CrossRefPubMedGoogle Scholar
  67. Sbarbati A, Bunnemann B, Cristofori P, Terron A, Chiamulera C, Merigo F et al (2002) Chronic nicotine treatment changes the axonal distribution of 68 kDa neurofilaments in the rat ventral tegmental area. Eur J Neurosci 16:877–882CrossRefPubMedGoogle Scholar
  68. Schmuck G, Kahl R (2009) The use of Fluoro-Jade in primary neuronal cell cultures. Arch Toxicol 83:397–403CrossRefPubMedGoogle Scholar
  69. Schmuck G, Ahr HJ, Schluter G (2000) Rat cortical neuron cultures: an in vitro model for differentiating mechanisms of chemically induced neurotoxicity. In Vitro Mol Toxicol 13:37–49Google Scholar
  70. Segura-Aguilara J, Kostrzewa RM (2004) Neurotoxins and neurotoxic species implicated in neurodegeneration. Neurotox Res 6:615–630CrossRefGoogle Scholar
  71. Sergent-Tanguy S, Chagneau C, Neveu I, Naveilhan P (2003) Fluorescent activated cell sorting (FACS): a rapid and reliable method to estimate the number of neurons in a mixed population. J Neurosci Meth 129:73–79CrossRefGoogle Scholar
  72. Shelton MK, McCarthy KD (2000) Hippocampal astrocytes exhibit Ca2+-elevating muscarinic cholinergic and histaminergic receptors in situ. J Neurochem 74:555–563CrossRefPubMedGoogle Scholar
  73. Shen C, Cheng XD, Li DH, Meng Q (2009) Investigation of rifampicin-induced hepatotoxicity in rat hepatocytes maintained in gel entrapment culture. Cell Biol Toxicol 25:265–274CrossRefPubMedGoogle Scholar
  74. Shi FD, Piao WF, Kuo YP, Campagnolo DI, Vollmer TL, Lukas RJ (2009) Nicotinic attenuation of central nervous system inflammation and autoimmunity. J Immunol 182:1730–1739CrossRefPubMedGoogle Scholar
  75. Tieu K, Ashe PC, Zuo DM, Yu PH (2001) Inhibition of 6-hydroxydopamine-induced p53 expression and survival of neuroblastoma cells following interaction with astrocytes. Neuroscience 103:125–132CrossRefPubMedGoogle Scholar
  76. Tornetta C, Gao ZY, Lee VM, Wof BA (1998) Regulation of amyloid precursor protein secretion by glutamate receptors in human Ntera 2 Neurons (NT2N)J. Biol Chem 273:14015–14021CrossRefGoogle Scholar
  77. Tsacopoulos M, Magistretti PJ (1996) Metabolic coupling between glia and neurons. J Neurosci 16:877–885PubMedGoogle Scholar
  78. Van Vliet E, Morath S, Eskes C, Linge J, Rappsilber J, Honegger P et al (2008) A novel in vitro metabolomics approach for neurotoxicity testing, proof of principle for methyl mercury chloride and caffeine. Neurotoxicology 29:1–12CrossRefPubMedGoogle Scholar
  79. Verderio C, Matteoli M (2001) ATP mediates calcium signaling between astrocytes and microglial cells: modulation by IFN-gamma. J Immunol 166:6383–6391PubMedGoogle Scholar
  80. Wang SJ (2007) Caffeine facilitation of glutamate release from rat cerebral cortex nerve terminals (synaptosomes) through activation protein kinase C pathway: an interaction with presynaptic adenosine A1 receptors. Synapse 61:401–411CrossRefPubMedGoogle Scholar
  81. Watts LT, Rathinam ML, Schenker S, Henderson GI (2005) Astrocytes protect neurons from ethanol-induced oxidative stress and apoptotic death. J Neurosci Res 80:655–666CrossRefPubMedGoogle Scholar
  82. Williams ES, Panko J, Paustenbach DJ (2009) The European Union’s REACH regulation: a review of its history and requirements. Critical Rev Toxicol 39:553–575CrossRefGoogle Scholar
  83. Woehrling EK, Hill EJ, Coleman MD (2007) Development of a neurotoxicity test-system, using human post-mitotic, astrocytic and neuronal cell lines in co-culture. Toxicol In Vitro 21:1241–1246CrossRefPubMedGoogle Scholar
  84. Woehrling EK, Hill EJ, Coleman MD (2010) Evaluation of the importance of astrocytes when screening for acute toxicity in neuronal cell systems. Neurotox Res 17:103–113CrossRefPubMedGoogle Scholar
  85. Wullner U, Seyfried J, Groscurth P, Beinroth S, Winter S, Gleichmann M et al (1999) Glutathione depletion and neuronal cell death: the role of reactive oxygen intermediates and mitochondrial function. Brain Res 826:53–62CrossRefPubMedGoogle Scholar
  86. Yi C, Xie KQ, Song FY, Yu LH, Zhao XL, Li GZ et al (2006) The changes of cytoskeletal proteins in plasma of acrylamide-induced rats. Neurochem Res 31:751–757CrossRefPubMedGoogle Scholar
  87. Yildiz D (2004) Nicotine, its metabolism and an overview of its biological effects. Toxicon 43:619–632CrossRefPubMedGoogle Scholar
  88. Yu PH, Zuo DM (1997) Enhanced tolerance of neuroblastoma cells towards the neurotoxin 6-hydroxydopamine following specific cell–cell interaction with primary astrocytes. Neuroscience 78:903–912CrossRefPubMedGoogle Scholar
  89. Yu SF, Zhao XL, Zhang TL, Yu LH, Li SX, Cui N et al (2005) Acrylamide-induced changes in the neurofilament protein of rat cerebrum fractions. Neurochem Res 30:1079–1085CrossRefPubMedGoogle Scholar
  90. Yu SF, Son FY, Yu JX, Zhao XL, Yu LH, Li GZ et al (2006) Acrylamide alters cytoskeletal protein level in rat sciatic nerves. Neurochem Res 31:1197–1204CrossRefPubMedGoogle Scholar
  91. Zhou JL, Liang JH, Zheng JW, Li CL (2004) Nerve growth factor protects R2 cells against neurotoxicity induced by methamphetamine. Toxicol Lett 150:221–227CrossRefPubMedGoogle Scholar
  92. Zimmer LA, Ennis M, Wiley RG, Shipley MT (1998) Nerve gas-induced seizures: role of acetylcholine in the rapid induction of Fos and glial fibrillary acidic protein in piriform cortex. J Neurosci 18:3897–3908PubMedGoogle Scholar
  93. Zurich MG, Honegger P, Shelter B, Costa LG, Monnet-Tschudi F (2004) Involvement of glial cells in the neurotoxicity of parathion and chlorpyrifos. Toxicol Appl Pharmacol 201:97–104CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • E. K. Woehrling
    • 1
    Email author
  • E. J. Hill
    • 1
  • E. E. Torr
    • 1
  • M. D. Coleman
    • 1
  1. 1.School of Life and Health SciencesAston UniversityBirminghamUK

Personalised recommendations