Oops They Did It Again! Carbon Nanotubes Hoax Scientists in Viability Assays

Abstract

New materials of emerging technological importance are single-walled carbon nanotubes (SWCNTs). Because SWCNTs will be used in commercial products in huge amounts, their effects on human health and the environment have been addressed in several studies. Inhalation studies in vivo and submerse applications in vitro have been described with diverging results. Why some indicate a strong cytotoxicity and some do not is what we report on here. Data from A549 cells incubated with carbon nanotubes fake a strong cytotoxic effect within the MTT assay after 24 h that reaches roughly 50%, whereas the same treatment with SWCNTs, but detection with WST-1, reveals no cytotoxicity. LDH, FACSassisted mitochondrial membrane potential determination, and Annexin-V/PI staining also reveal no cytotocicity. SWCNTs appear to interact with some tetrazolium salts such as MTT but not with others (such as WST-1, INT, XTT). This interference does not seem to affect the enzymatic reaction but lies rather in the insoluble nature of MTT−formazan. Our findings strongly suggest verifying cytotoxicity data with at least two or more independent test systems for this new class of materials (nanomaterials). Moreover, we intensely recommend standardizing nanotoxicological assays with regard to the material used: there is a clear need for reference materials. MTT−formazan crystals formed in the MTT reaction are lumped with nanotubes and offer a potential mechanism to guide bioremediation and clearance for SWCNTs from "contaminated" tissue. SWCNTs are good supporting materials for tissue growth, as attachment of focal adhesions and connections to the cytoskeleton suggest.

Low Doses of Pristine and Oxidized Single-Wall Carbon Nanotubes Affect Mammalian Embryonic Development

“…Due to the production of water-soluble formazan salts, such test has been proven compatible with the use of SWCNTs. 42 For mES cell differentiation into embryoid bodies the "hanging drops" protocol was followed according to ARTICLE the EST guidelines. On day 10 the presence of spontaneous contraction of the embryoid bodies was visually evaluated under the light microscope.…”

High throughput toxicity screening and intracellular detection of nanomaterials

“…Assays for cellular metabolic activity, oxidative stress evaluation, apoptosis detection, and cell membrane integrity49, 50, 51, 52, 53, 54, 55 have been developed for analyzing cytotoxicity of chemicals and these are widely used also in NM‐induced cytotoxicity screening. The methods are time‐consuming, labor‐intensive, complex, and in some instances unreliable owing to NM interferences 53, 56, 57.…”

“…The methods are time‐consuming, labor‐intensive, complex, and in some instances unreliable owing to NM interferences 53, 56, 57. The reagents employed in such methods may interact with some of the tested NMs or may interfere with spectrophotometric readings, leading to unreliable results 49, 50, 55. Conventional assays focus on specific endpoints, without providing information about the dynamic biological processes leading to those endpoints, or the specific time points and concentrations at which different toxic effects are induced by NMs 54, 58.…”

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

“…Secondly, the nanomaterials could interferet hrough binding with the formazanc rystals formed, therebyg enerating af alse negative result and cause an underestimation in the number of viable cells. [41,[57][58][59][60][61][62] To investigate whether the test materials could induce ar eduction of the active tetrazolium salt, different concentrationso ft he Group 5t ransition metal ditelluride was exposed with the MTT assay in ac ell free environment. To study the probable binding effect, ascorbic acid was added to different concentrations of MTe 2 to induce the formazan crystals formation from the MTT assay.I fM Te 2 binds with the formazan crystals, one would expect to see ad ecreasei nt he percentage of formazan with increasing amount of the test materials.…”