Breznan Dalibor, Das Dharani, MacKinnon-Roy Christine, Simard Benoit, Kumarathasan Premkumari, Vincent Renaud
Inhalation Toxicology Laboratory, Hazard Identification Division, EHSRB, Research and Radiation Directorate, HECSB, Health Canada, Ottawa, ON K1A 0K9, Canada.
Analytical Biochemistry and Proteomics Laboratory, Mechanistic Studies Division, EHSRB, Research and Radiation Directorate, HECSB, Health Canada, Ottawa, ON K1A 0K9, Canada.
Toxicol In Vitro. 2015 Feb;29(1):142-7. doi: 10.1016/j.tiv.2014.09.009. Epub 2014 Oct 2.
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.
体外细胞毒性测定是筛选工程纳米材料（NM）细胞毒性的重要工具。刃天青活细胞测定法被广泛使用，因为它是非破坏性的，非常适合高通量平台。然而，纳米材料，特别是碳纳米管（CNT），可能会通过淬灭透射光或荧光干扰测定。我们表明，使用刃天青测定法并对澄清的上清液进行时间点读数可以解决这个问题。将人肺上皮（A549）和小鼠巨噬细胞（J774A.1）细胞系在96孔板中以每孔200μL培养基暴露于纳米材料。孵育24小时后，取出100μL上清液，用培养基中的刃天青试剂替换，将10分钟和120分钟时的等分试样转移到黑色壁96孔板中。将板快速离心以沉淀残留的碳纳米管，并对荧光进行顶部读数（λEx = 540nm，λEm = 600nm）。该程序已针对碳纳米管以及二氧化硅纳米颗粒（SiNP）进行了验证。没有迹象表明碳纳米管会使刃天青减少。然后评估刃天青还原的荧光产物试卤灵的稳定性。我们发现极性碳纳米管可能会降低试卤灵的荧光信号，可能是通过氧化为刃天青或过度还原为羟基试卤灵。这种影响可以很容易地量化以消除偏差。我们建议，对于光学/化学活性纳米材料的荧光/比色体外毒理学评估，必须仔细考虑，以消除由于纳米材料本身引起的任何潜在假象。
