Manshian, B Jenkins, GJS Barron, A Wright, CJ Williams, P Doak, SH
Introduction: Nanotechnology is a fast growing industry with nanomaterials already applied in over 800 consumer products. Single walled carbon nanotubes (SWCNTs) are a popular group of nanomaterials due to their beneficial properties, but they are also the cause of great concern due to their fibrous structure and high aspect ratio. Amid a fast growing industry there is still the lack of sufficient understanding of their role and the mechanisms involved in DNA damage. Therefore, this study examines these parameters taking into consideration the effects of geometry and the surface characteristics of the nanotubes.
Methods: A range of techniques were utilised to characterise the physico-chemical features of the test materials (SWCNT with a manufactured length of 400-800nm, 1-3m and 5-30m) to determine purity, morphology surface area, surface charge, agglomerate size distribution and cellular uptake. BEAS-2B human bronchial epithelial cells and MCL5 human lymphoblastoid B cells were treated with SWCNTs for 24 or 48 hrs. The genotoxic and mutagenic potential of these nanomaterials were determined using the CBMN cytokinesis blocked micronucleus assay and the hprt forward mutation assay. Oxidative stress was also assessed using the DCFH-DA fluorescence assay within cells and in cell-free solution; catalase assay, and oxidative damage pathway gene expression profiling by RT-PCR.
Results: Physico-chemical analysis revealed that after purification the SWCNTs were 97% pure. Under test conditions, they were mainly found to exist in small spherical agglomerates and as bundles of parallel aligned tubes. When applied to BEAS-2B and MCL5 cells, significant increases in micronucleus frequency (and therefore chromosomal damage) were encountered in a concentration and time-dependent manner in both cell types in the absence of cytotoxicity. Interestingly, in both cell types exactly the same order of potency for the induction of gross chromosomal damage was observed, 400-800nm > 5-30µm ≥ 1-3µm. Over the same dose-range SWCNTs only gave rise to significant increases in hprt point mutation levels at the highest 100g/ml dose with the 400-800nm and 1-3µm SWCNT sample, although the latter was far more potent. The oxidative stress assays demonstrated the involvement of reactive oxygen species in the genotoxicity induced by these nanomaterials.
Conclusions: This study demonstrates that SWCNTs induce significant levels of chromosomal damage at sub-cytotoxic concentrations. Additionally, the hprt assay demonstrated that 1-3m SWCNTs are mutagenic in mammalian cells at high doses which might have a bearing on their possible carcinogenicity. Furthermore, it is highly possible that this observed genotoxicity is induced by an oxidative stress based mechanism.