title: Non-specific interaction of carbon nanotubes with the resazurin assay reagent: impact on in vitro assessment of nanoparticle cytotoxicity.
abstract: In vitro cytotoxicity assays are essential tools in the screening of engineered nanomaterials (NM) for cellular toxicity. The resazurin live cell assay is widely used because it is non-destructive and is well suited for high-throughput platforms. However, NMs, in particular carbon nanotubes (CNT) can interfere in assays through quenching of transmitted light or fluorescence. We show that using the resazurin assay with time-point reading of clarified supernatants resolves this problem. Human lung epithelial (A549) and murine macrophage (J774A.1) cell lines were exposed to NMs in 96-well plates in 200 μL of media/well. After 24 h incubation, 100 μL of supernatant was removed, replaced with resazurin reagent in culture media and aliquots at 10 min and 120 min were transferred to black-wall 96-well plates. The plates were quick-spun to sediment the residual CNTs and fluorescence was top-read (λEx=540 nm, λEm=600 nm). The procedure was validated for CNTs as well as silica nanoparticles (SiNP). There was no indication of reduction of resazurin by the CNTs. Stability of resorufin, the fluorescent product of the resazurin reduction was then assessed. We found that polar CNTs could decrease the fluorescence signal for resorufin, possibly through oxidation to resazurin or hyper-reduction to hydroxyresorufin. This effect can be easily quantified for elimination of the bias. We recommend that careful consideration must be given to fluorimetric/colorimetric in vitro toxicological assessments of optically/chemically active NMs in order to relieve any potential artifacts due to the NMs themselves.
authors.affiliation: Inhalation Toxicology Laboratory, Hazard Identification Division, EHSRB, Research and Radiation Directorate, HECSB, Health Canada, Ottawa, ON K1A 0K9, Canada. Electronic address: Dalibor.Breznan@hc-sc.gc.ca.
authors.forename: Dalibor
authors.initials: D
authors.lastname: Breznan
authors.affiliation: Analytical Biochemistry and Proteomics Laboratory, Mechanistic Studies Division, EHSRB, Research and Radiation Directorate, HECSB, Health Canada, Ottawa, ON K1A 0K9, Canada.
authors.forename: Dharani
authors.initials: D
authors.lastname: Das
authors.affiliation: Inhalation Toxicology Laboratory, Hazard Identification Division, EHSRB, Research and Radiation Directorate, HECSB, Health Canada, Ottawa, ON K1A 0K9, Canada.
authors.forename: Christine
authors.initials: C
authors.lastname: MacKinnon-Roy
authors.affiliation: NRC Steacie Institute for Molecular Sciences, Ottawa, ON K1A 0R6, Canada.
authors.forename: Benoit
authors.initials: B
authors.lastname: Simard
authors.affiliation: Analytical Biochemistry and Proteomics Laboratory, Mechanistic Studies Division, EHSRB, Research and Radiation Directorate, HECSB, Health Canada, Ottawa, ON K1A 0K9, Canada.
authors.forename: Premkumari
authors.initials: P
authors.lastname: Kumarathasan
authors.affiliation: Inhalation Toxicology Laboratory, Hazard Identification Division, EHSRB, Research and Radiation Directorate, HECSB, Health Canada, Ottawa, ON K1A 0K9, Canada.
authors.forename: Renaud
authors.initials: R
authors.lastname: Vincent
mesh_terms: D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D005453:Fluorescence; D006801:Humans; D053758:Nanoparticles / Q000633:toxicity*; D037742:Nanotubes, Carbon / Q000009:adverse effects*; D010078:Oxazines; D018675:Toxicity Tests / Q000379:methods*; D014966:Xanthenes
chemical_list: D037742:Nanotubes, Carbon; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin
keywords: Carbon nanotubes; Cell viability; Cytotoxicity; In vitro; Interaction; Resazurin