Confounding experimental considerations in nanogenotoxicology

Abstract

The development of novel nanomaterials with unique physico-chemical properties is increasing at a rapid rate, with potential applications across a broad range of manufacturing industries and consumer products. Nanomaterial safety is therefore becoming an increasingly contentious issue that has intensified over the past 4 years, and in response, a steady stream of studies focusing on nanotoxicology are emerging. However, it is becoming increasingly evident that nanomaterials cannot be treated in the same manner as chemical compounds with regards to their safety assessment, as their unique physico-chemical properties are also responsible for unexpected interactions with experimental components that generate misleading data-sets. In this report, we focus on nanomaterial interactions with colorimetric and fluorometric dyes, components of cell culture growth medium and genotoxicity assay components, and the resultant consequences on test systems are demonstrated. Thus, highlighting some of the potential confounding factors that need to be considered in order to ensure that in vitro genotoxicity assays report true biological impacts in response to nanomaterial exposure.

Single-walled carbon nanotube-induced mitotic disruption

“…Fluorescence in situ hybridization (FISH) for human chromosomes 1 and 4 was used to determine the chromosome number (Abbott Molecular, Des Plaines, IL) according to the guidelines of the American College of Medical Genetics [43]. To yield binucleate cells to indicate any nondisjunction of chromosomes 1 and 4, cytochalasin B was considered, however; use of this compound inhibits uptake of carbon nanotubes by endocytosis [21]. A minimum of 100 interphase cells of good FISH morphology were analyzed to determine the number of chromosome 1 and 4.…”

“…In vitro investigations have shown SWCNT-induced DNA damage in established cancer cell lines, immortalized bronchial epithelial cells as well as primary mouse embryo fibroblasts and human mesothelial cells [18][19][20]. Micronuclei have been observed in significant numbers following in vitro treatment with SWCNT or MWCNT indicating disruption of the mitotic spindle apparatus [19,21]. The presence of chromosome centromeres in the micronuclei indicates the loss of whole chromosomes.…”

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

“…[57][58][59] In order to check for anyi nterference, ac ontrol experiment was performed by incubating varying concentrations of test materials with WST-8 assay for an hour in ac ell free condition to check for possible formazan production through material-inducedr eductions. Figure 4d etails the dataoft he WST-8 control experiments.…”

“…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.…”

Important issues in the cytotoxicity screening of nano-sized materials

“…Examples for interference caused by NMs comprise conversion of tetrazolium salts ( [5][6][7]), adsorption of dyes ( [8,9]), interference by absorbance ( [10,11]), by fluorescence ( [9,12]), by binding of proteins ( [13]), by dye degradation ( [14]), by redox reactions ( [15]) and by light scattering ( [16,17]). …”