Interference of engineered nanoparticles with in vitro toxicity assays

Abstract

Accurate in vitro assessment of nanoparticle cytotoxicity requires a careful selection of the test systems. Due to high adsorption capacity and optical activity, engineered nanoparticles are highly potential in influencing classical cytotoxicity assays. Here, four common in vitro assays for oxidative stress, cell viability, cell death and inflammatory cytokine production (DCF, MTT, LDH and IL-8 ELISA) were assessed for validity using 24 well-characterized engineered nanoparticles. For all nanoparticles, the possible interference with the optical detection methods, the ability to convert the substrates, the influence on enzymatic activity and the potential to bind proinflammatory cytokines were analyzed in detail. Results varied considerably depending on the assay system used. All nanoparticles tested were found to interfere with the optical measurement at concentrations of 50 μg cm⁻² and above when DCF, MTT and LDH assays were performed. Except for Carbon Black, particle interference could be prevented by altering assay protocols and lowering particle concentrations to 10 μg cm⁻². Carbon Black was also found to oxidize H₂DCF-DA in a cell-free system, whereas only ZnO nanoparticles significantly decreased LDH activity. A dramatic loss of immunoreactive IL-8 was observed for only one of the three TiO₂ particle types tested. Our results demonstrate that engineered nanoparticles interfere with classic cytotoxicity assays in a highly concentration-, particle- and assay-specific manner. These findings strongly suggest that each in vitro test system has to be evaluated for each single nanoparticle type to accurately assess the nanoparticle toxicity.

Assessing nanoparticle toxicity in cell-based assays: influence of cell culture parameters and optimized models for bridging the in vitro–in vivo gap

“…Obviously NP with optical properties might alter the outcome of an assay based on a spectrophotometric or fluorometric read-out 33 32 . These data underscore the importance of validating the assays for each specific type of NP.…”

“…It is crucial to view nanotoxicology as a distinct category of toxicology since it has been observed that standard toxicity assays, which were initially developed for the evaluation of pharmacological agents, are often inadequate for nanotoxicity assessment. This can be attributed to the different mechanisms through which NP may evoke toxic effects, the specific behaviour of the NP in culture media and the possible interference of NP with various in vitro and in vivo toxicity assays 9,[32][33][34] .…”

Cytotoxicity of Group 5 Transition Metal Ditellurides (MTe2; M=V, Nb, Ta)

“…[39][40][41] As such, control experiments were performed in the absence of cells to check for possible sources of particle-induced interferences,t od etermine the suitability of the assays. Therefore, we set out to determinep ossible forms of particle-inducedi nterferences for two commonly used assays which have similar working mechanisms:W ST-8 and methyl thiazolyldiphenyl-tetrazoliumbromide (MTT) assays.…”

“…[10,35] The remaining cell viability of the A549 cellsa fter 24 hi ncubation with variousM Te 2 concentrations was measured and analysed using the water-soluble tetrazolium salt (WST-8) assay.F urthermore, controle xperiments were also conducted in the absence of cells to check for possible sources of particle-induced interferences between MTe 2 with cell viability assaym arkersw hichc ould cause false positive or false negative resultstob eproduced. [39][40][41] Results and Discussion…”

Testing Nanomaterial Toxicity in Unicellular Eukaryotic Algae and Fish Cell Lines

“…For example, carbon-based NMs have a high sorption capacity also for hydrophobic fl uorescence indicator dyes, which may render some of these dyes impossible to use to measure NM cellular effects [ 52 ]. Another important aspect is how to express the effect assessment results in terms of exposure or dose.…”