POTENTIAL GENOTOXIC RISKS OF EXPOSURE OF HUMAN CELLS TO SINGLE WALLED CARBON NANOTUBES OF VARYING LENGTHS
Manshian, Bella Jenkins, Gareth JS Barron, Andy Wright, Chris Doak, Shareen H
Carbon nanotubes (CNT) are a major group of nanomaterials increasingly used in engineering, electronics, environmental remediation and medical healthcare. However, information on the toxicity of these nanomaterials is still fragmentary with a lack of genotoxicity studies. In-vitro and in-vivo toxic responses elicited in CNTs have been attributed to the presence of metal contaminants, length and surface area, or degree of oxidation. In this study, the structural properties of SWCNT were considered in relation to their cytotoxic and genotoxic effects in two human cell lines representing the circulatory (MCL5) and respiratory (BEAS-2B) systems. These were compared to an established carcinogen, crocidolite asbestos, due to structural similarities in terms of fibrous nature with a high aspect ratio.
Following extensive physico-chemical analysis of the SWCNTs (400-800nm, 1-3um and 5-30um lengths) BEAS-2B (bronchial epithelial) and MCL5 (lymphoblastoid B) cells were treated with SWCNTs for 24 or 48hrs. The genotoxic and mutagenic potential of these nanomaterials were determined using the CBMN and the hprt forward mutation assays. Oxidative stress was assessed using the DCFH-DA fluorescence assay within cells and in cell-free systems. These results were confirmed with the catalase colorimetric assay and oxidative damage pathway gene expression profiling by RT-PCR.
Under test conditions, the SWCNTs were mainly found to exist in small spherical agglomerates and in the 1-3um sample also as bundles of parallel aligned tubes. When applied to cell cultures, significant increases in micronucleus (MN) frequency, therefore chromosomal damage, were encountered in a concentration and time-dependent manner in the absence of cytotoxicity. Both cell types presented with the same order of MN induction; 400-800nm > 5-30um > 1-3um. These results were comparative to crocidolite asbestos. SWCNTs gave rise to significant increases in point mutations at 100ug/ml dose in the 400-800nm and highly significant levels at doses 25ug/ml in the 1-3um sample. A threshold effect was observed in the 1-3um CNTs using the Hockey stick statistical analysis. Kinetochore fluorescent probes (CREST) applied to the treated BEAS-2B cells showed centromere negative MNi indicating clastogenicity. All three tests for oxidative damage demonstrated a strong correlation between the genotoxic damage observed mainly in the 1-3um sample. This increase was substantially reduced when the antioxidant N-Acetyl Cysteine was added. Overall, this study provides the first evidence of the potential of SWCNTs to induce mutagenic and genotoxic events.