Single-cell ELISA and flow cytometry as methods for highlighting potential neuronal and astrocytic toxicant specificity

Elizabeth Woehrling, Eric J. Hill, E.E. Torr, Mike Coleman

Research output: Contribution to journalArticle

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.
LanguageEnglish
Pages472-483
Number of pages12
JournalNeurotoxicity Research
Volume19
Issue number3
Early online date15 Jun 2010
DOIs
Publication statusPublished - Apr 2011

Fingerprint

Flow cytometry
Glial Fibrillary Acidic Protein
Intermediate Filaments
Flow Cytometry
Cytotoxicity
Enzyme-Linked Immunosorbent Assay
Chloroquine
Chloramphenicol
Nicotine
Atropine
Toxicity
Screening
Acrylamide
Poisons
Neurology
Cell death
Rifampin
Verapamil
Caffeine
Neurons

Keywords

  • astrocytes
  • tumor cell line
  • cell survival
  • cytoskeleton
  • enzyme-linked immunosorbent assay
  • flow cytometry
  • glial fibrillary acidic protein
  • humans
  • neurons
  • neurotoxins

Cite this

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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.",
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Single-cell ELISA and flow cytometry as methods for highlighting potential neuronal and astrocytic toxicant specificity. / Woehrling, Elizabeth; J. Hill, Eric; Torr, E.E.; Coleman, Mike.

In: Neurotoxicity Research, Vol. 19, No. 3, 04.2011, p. 472-483.

Research output: Contribution to journalArticle

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