Molecular Characterization of the Cytotoxic Mechanism of Multiwall Carbon Nanotubes and Nano-Onions on Human Skin Fibroblast

Abstract

The increasing use of nanotechnology in consumer products and medical applications underlies the importance of understanding its potential toxic effects to people and the environment. Although both fullerene and carbon nanotubes have been demonstrated to accumulate to cytotoxic levels within organs of various animal models and cell types and carbon nanomaterials have been exploited for cancer therapies, the molecular and cellular mechanisms for cytotoxicity of this class of nanomaterial are not yet fully apparent. To address this question, we have performed whole genome expression array analysis and high content image analysis based phenotypic measurements on human skin fibroblast cell populations exposed to multiwall carbon nano-onions (MWCNOs) and multiwall carbon nanotubes (MWCNTs). Here we demonstrate that exposing cells to MWCNOs and MWCNTs at cytotoxic doses induces cell cycle arrest and increases apoptosis/necrosis. Expression array analysis indicates that multiple cellular pathways are perturbed after exposure to these nanomaterials at these doses, with material-specific toxigenomic profiles observed. Moreover, there are also distinct qualitative and quantitative differences in gene expression profiles, with each material at different dosage levels (6 and 0.6 microg/mL for MWCNO and 0.6 and 0.06 microg/mL for MWCNT). MWCNO and MWCNT exposure activates genes involved in cellular transport, metabolism, cell cycle regulation, and stress response. MWCNTs induce genes indicative of a strong immune and inflammatory response within skin fibroblasts, while MWCNO changes are concentrated in genes induced in response to external stimuli. Promoter analysis of the microarray results demonstrate that interferon and p38/ERK-MAPK cascades are critical pathway components in the induced signal transduction contributing to the more adverse effects observed upon exposure to MWCNTs as compared to MWCNOs.

Cellular Effect of High Doses of Silica-Coated Quantum Dot Profiled with High Throughput Gene Expression Analysis and High Content Cellomics Measurements

“…nanotubes and nanoonions, in these cell lines. 27 These observations are also consistent with gene expression results presented below.…”

“…In addition, previous studies have been done using these types of model systems, allowing us to directly compare our results to results from these studies. 27 These results demonstrate that the surface chemistry of Qdots is very important for determining toxicity and further open the field for long-term labeling of live cells and in vivo clinical imaging applications. This should provide guidance for any future improvements upon surface chemistry to reduce or eliminate the toxicity of other nanomaterials as well.…”

“…This is in strong contrast to treatment of these same cells with carbon nanotubes and nano-onions. 27 For instance, the MWCNT at a concentration of 0.6 mg/L induced significant changes in 216 genes, while the PEG-silaneQdot induced changes in 20 times fewer genes at a much higher concentration of 40 mg/L (80 nM, with molecular weight approximately 500 KDa). Because there is little to no toxicity for these PEG-silane-Qdots compared to other types of nanoparticles, this provides evidence that the composition and surface functionalization of the particle are the most important determinants of toxicity.…”

“…In addition, genotoxicity data of carbon nanotubes and nano-onions are available for these cells and can be used for comparison purposes. 27 Human skin fibroblast and lung fibroblast cells exposed to 8 or 80 nM PEG-silane-Qdots for 48 h internalize them ( Figure 1B). As shown in Figure 1B, all cells are labeled by Qdots.…”

“…First, we studied the proliferation profile of this cell line by incorporating BrdUrd into replicating DNA and counterstaining with PI to determine total DNA content. 27 For each individual HSF-42 cell, the ratio of the signal intensity from antibody staining of incorporated BrdUrd versus total DNA content measured by PI staining is plotted in a scatter plot. We analyzed the cell cycle status of more than 20,000 cells and then classified them into G0/G1, S, or G2/M phases.…”