title: In vitro assessment of cobalt oxide particle toxicity: identifying and circumventing interference.
abstract: The continuing development of nanotechnology necessitates the reliable assessment of potential adverse health consequences associated with human exposures. The physicochemical properties of nanomaterials can be responsible for unexpected interactions with components of classical toxicity assays, which may generate erroneous interpretations. In this paper, we describe how particle interference can be observed in in vitro toxicity tests (CellTiter Blue, CyQUANT, WST-1 and CellTiter-Glo assay) and in cell biology tests using flow cytometry (cell cycle analysis). We used cobalt oxide (Co3O4) particles as an example, but these assays can be performed, in principle, regardless of the nanoparticle considered. We have shown that cobalt particles interfere with most of these tests. We adapted the protocol of the CellTiter-Glo assay to circumvent this interference and demonstrated that, using this protocol, the toxicity level is consistent with results obtained using the clonogenic assay, which is considered to be the reference test. Before assessing particle toxicity using in vitro toxicity tests, interference testing should be performed to avoid false interpretations. Furthermore, in some cases of interference, protocol adaptation can be considered to allow the reliable use of these quick and convenient in vitro tests.
authors.affiliation: CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze F-30207, France.
authors.forename: Carine
authors.initials: C
authors.lastname: Darolles
authors.forename: Nicole
authors.initials: N
authors.lastname: Sage
authors.forename: Jean
authors.initials: J
authors.lastname: Armengaud
authors.forename: Véronique
authors.initials: V
authors.lastname: Malard
mesh_terms: D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors; D001681:Biological Assay / Q000379:methods*; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003035:Cobalt / Q000737:chemistry / Q000633:toxicity*; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry / Q000633:toxicity* / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D010087:Oxides / Q000737:chemistry / Q000633:toxicity*; D010316:Particle Size; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D010087:Oxides; D003035:Cobalt; D000255:Adenosine Triphosphate; C060728:cobalt oxide
keywords: Cobalt; In vitro methods; Interference; Particles; Toxicity; Viability assays