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Stable Colloidal Dispersions of C60 Fullerenes in Water: Evidence for Genotoxicity

Alok Dhawan1, Julian S. Taurozzi2, Alok K. Pandey3, Wenqian Shan2, Sarah M. Miller4, Syed A. Hashsham5, and Volodymyr V. Tarabara6. (1) Developmental Toxicology Division / Department of Civil and Environmental Engineering, Industrial Toxicology Research Centre / Michigan State University, P. O. Box 80, Mahatma Gandhi Marg, Lucknow, India, (2) Department of Civil and Environmental Engineering, Michigan State University, A110 Research Complex Engineering, East Lansing, MI 48824, (3) Developmental Toxicology Division, Industrial Toxicology Research Centre, P. O. Box 80, Mahatma Gandhi Marg, Lucknow, India, (4) Department of Civil and Environmentail Engineering, Michigan State University, A127 Research Complex Engineering, East Lansing, MI 48824, (5) Department of Civil and Environmental Engineering / The Center for Microbial Ecology, Michigan State University, East Lansing, MI 48824, (6) Civil and Environmental Engineering, Michigan State University, A132 Research Complex Engineering, East Lansing, MI 48824

Concerns regarding potential health risks and the environmental impact of engineered nanomaterials prompt a proactive approach to ensuring that the burgeoning nanotech industry is environmentally benign and sustainable (1-3). One salient example of a novel carbon-based nanoscale material of growing practical importance is C60 fullerene. C60 had been thought to exist in water only in molecular solutions of very low concentrations (less than 10-9 mg/L (4)) unless its surface was derivatized to render C60 more hydrophilic. However, it was shown that under certain conditions, pristine C60 can form suspensions of C60 clusters (nC60) in water (5,6). One implication of these findings is that fullerene hydrosols are stable and nC60 particles are likely to persist in the aqueous environment.

To evaluate the environmental impact of waterborne fullerenes, both their physicochemical properties and toxicity need to be assessed. Several important physicochemical characteristics of nC60 have been shown to strongly depend on nC60 preparation technique (7). Techniques that have been developed to produce stable dispersions of fullerenes in water can be grouped into two categories: (i) methods based on solvent exchange wherein solution of fullerenes in an organic solvent is mixed with water and the organic solvent is then removed from the mixture and (ii) methods based on direct dispersion of powdered C60 in water followed by prolonged mixing of the dispersion. In an earlier study, nC60 prepared by solvent exchange using benzene-THF-acetone was found to have no effect on the proliferation rate of keratinocytes or fibroblasts (5). Several other studies on the cytotoxicity of nC60 have been published recently. Fortner at al. (8) found that the growth of both E.coli and Bacillus subtilis were inhibited by THF/nC60 at a concentration of 0.4 mg/L. For THF/nC60, Sayes at al. determined LC50 for human skin cells to be 20 mg/L (9). There is evidence, however, of the presence of residual THF (7,8,10) most likely associated with nC60 particles either as adsorbed species or intercalated into the bulk of the nC60 cluster (11); depending on the residual concentration, the solvent may interfere with toxicity measurements.

To the best of our knowledge, no data has been published on whether nC60 also damages DNA. Based on the observations that nC60: i) is capable of producing reactive oxygen species (12), ii) causes leaky cytoplasmic membrane (12), and iii) may include molecular fullerene C60@{H2O}m as a component of nC60 hydrosol (13,14), it is hypothesized that nC60 will also result in DNA damage. Therefore, the study was undertaken to test the above hypothesis, i.e., to correlate DNA damage to the presence of nC60. Because the presence of organic solvent in nC60 suspensions may confound toxicity data, this study used preparation methods that were either free of organic solvent (extended mixing method) or employed solvent exchange method with ethanol used as the solvent, which is known to be non-genotoxic at the concentrations used (15).

The effect of nC60 on DNA damage was studied using the single cell gel electrophoresis assay, also known as Comet assay (16,17). Our study demonstrated a strong correlation between the presence of nC60 and DNA damage and to our best knowledge, the results represent the first toxicity data for colloidal fullerenes produced by simple mixing in water and first genotoxicity data for aqueous nC60 colloids. An analysis of cell distribution with varying degree of DNA damage shows that both increasing the dose of nC60 and increasing the exposure time cause an increase in cell damage. nC60 suspensions produced by extended mixing elicited higher genotoxic response for the same nC60 concentration. Oxygen radical- and electron transfer-mediated DNA damage by nC60 and hydrated molecular C60 were hypothesized to be possible reasons for genotoxicity.

We attribute this genotoxic response to the exposure to nC60 to one of or a combination of the following three reasons:

1) nC60 produces oxygen radicals and causes leaky cytoplasmic membranes. Both molecular C60 and nC60 produce oxygen radicals, which have been shown to cause lipid peroxidation in three different types of human cell lines (12). There is evidence that nC60 can also cause "leaky" cytoplasmic membranes suggesting that oxygen radical, molecular C60, and nC60 colloids may all have access to internal cellular organelles.

2) nC60 may include molecular hydrated C60@{H2O}m fraction. Using molecular models, Zhao et al (2005) demonstrated that the binding energy of two C60 molecules in aqueous solution is -7.5 kcal/mol while the binding energy between a 20 ns long oligonucleotide and C60 is between -27 to -42 kcal/mol. This binding energy is in the same range as the binding energy for signature oligonucleotides probes specifically designed to hybridize with their target sequences (-16 to -85 kcal/mol; (18)). Thus, a “partitioning” of C60 from aqueous solution into DNA matrix or other organic matrices (if present) is also possible.

3) Pristine and modified C60 cause DNA damage. Using an oligonucleotide attached to C60 carboxylic acid, Tokuyama et al. showed that upon photoactivation, C60 carboxylic acid cuts at guanine sites in a DNA sequence (19). Later, Boutourine et al. confirmed that guanine sites in the vicinity of C60 are preferentially cut and presumed that this may be due to the effect of oxygen radical on DNA damage (20). This characteristic of pristine C60 has also been implicated in virus inactivation (21) and in nonenzymatic cleavage of DNA (22), among others. Evidence is also available for an alternative mechanism (i.e., not implicating oxygen radical) of DNA damage due to electron transfer between C60 and oligonucleotides. Pristine C60 is capable of accepting up to 6 electrons (23).

References

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