Single-cell ELISA and flow cytometry as methods for highlighting potential neuronal and astrocytic toxicant specificity.
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.
Woehrling
E K
EK
School of Life and Health Sciences, Aston University, Aston Street, Birmingham, UK. e.k.woehrling@aston.ac.uk
Hill
E J
EJ
Torr
E E
EE
Coleman
M D
MD
D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D006801:Humans; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009498:Neurotoxins / Q000633:toxicity*
D005904:Glial Fibrillary Acidic Protein; D009498:Neurotoxins