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Nanotoxicology

Signs of stress

Nature Nanotechnologyvolume 1pages2324 (2006) | Download Citation

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The rapid expansion of nanotechnology has resulted in a vast array of nanoparticles that vary in size, shape, charge, chemistry, coating and solubility. Take carbon nanotubes, for example, which have been intensively studied because they have new and unusual mechanical, electronic and other properties. The potential toxicity of these materials has attracted attention because of their apparent similarities to asbestos and other carcinogenic fibres (Fig. 1). Carbon nanotubes are long, thin (just nanometres in diameter) and insoluble — all factors that contribute to fibre toxicity in the lungs1. A study by Andre Nel of the University of California, Los Angeles and co-workers now suggests that the hazards are best predicted by examining which nanomaterials cause most oxidative injury within cells2.

Figure 1: A rat lung cell attempting to ingest carbon nanotubes that are longer than the distance that the cell can stretch, which means that the rat cannot remove such nanotubes from the body.
Figure 1

This optical microscopy image is superimposed with confocal images of the protein cytoskeleton that gives the cell structure and its ability to move. F-actin is shown in red; tubulin in green. (Image provided by D. Brown, Napier Univ. and I. Kinloch, Univ. Manchester).

However, when testing the toxicity induced by carbon nanotubes, should we consider single- or multiwalled tubes? Long or short nanotubes? How long or short? Should we remove metal catalysts? Do we use functionalized or non-functionalized particles? Should we use pristine tubes or should they be tested in the form in which they might actually be used? The list of variations is endless and poses a real problem for toxicologists.

However, the challenge for toxicologists is not to test every variation of a new nanoparticle generated but, instead, to identify key factors or tests that can be used to predict toxicity, permit targeted screening and allow materials scientists to generate new, safer nanoparticles with this structure-toxicity information in mind.

Nel and co-workers have now taken a major step in this direction by systematically studying how nanomaterials can induce oxidative injury within cells2. The basic idea is that if we know how this process occurs, we can begin to compare the toxic potential of different nanomaterials by looking at the way they generate tiny reactive molecules in the cell.

Oxidative stress is caused by an imbalance between damaging oxidants and protective antioxidants. This state of affairs can impair or destroy cell proteins, lipids and DNA, leading to the deterioration in cell function or toxicity. Oxidants include hydrogen peroxide and hydroxyl radicals (collectively known as reactive oxygen species or ROS); antioxidants are molecules such as vitamin C and glutathione that scavenge the unwanted oxidants. There is abundant evidence to show that ambient pollution particles in the atmosphere3 such as diesel soot, bulk nanoparticles such as carbon black4, and engineered nanoparticles such as fullerenes can generate reactive oxygen species and oxidative stress5,6,7. But if there is already plenty of evidence, why is the work of Nel and colleagues important?

Our current knowledge of the toxicology of a variety of particles including environmental nanoparticles and various nanoparticles that have been manufactured in bulk for decades (for example, titanium dioxide) shows a clear link between oxidative stress and diseases including cancer, asthma and cardiovascular ailments. We can therefore compare the oxidative stress profiles of well-studied materials with newly engineered nanomaterials and, it is hoped, extrapolate the data to yet newer materials. Furthermore, oxidative stress may be a useful criterion to pinpoint which physical factors are associated with the biological insults. If carefully thought out, such studies could serve as important building blocks towards the development of more-efficient screening strategies for new nanomaterials.

Nel and colleagues examined a sample of ambient ultrafine particles and four types of manufactured nanoparticles — carbon black, titanium dioxide, fullerol and polystyrene spheres — and compared how they generated small reactive molecules and induced cell injury. Through a series of assays, Nel and colleagues found that ambient particles and positively charged polystyrene beads generated high levels of reactive molecules and induced oxidative stress in lung macrophages (defence cells in our body that gobble up particulate matter). Little activity was observed for carbon black, TiO2 and negatively charged polystyrene beads. Nanoparticles with different chemical composition, charge and size clearly induced different levels of cell injury. Nel and colleagues propose that oxidative stress is a suitable measure for comparing and discriminating the toxic effects of different nanoparticles.

Although the work is convincing, there are factors that still need to be explored. First, the work focused on only one nanoparticle concentration (10 μg ml−1). Examining a range of concentrations may provide further information on the relative potency of these different particle types.

Second, the cell assays used only one cell type. The rat cells used in the study have been engineered to reproduce indefinitely. Although such cell lines are commonly used in toxicity studies, using macrophages taken directly from humans will help to ascertain if the cell culture model is accurate. One of the main functions of macrophages is to generate reactive molecules as they ingest foreign particles, so it is possible that they will be more resistant towards oxidative stress than other cell types. Comparing the profiles for cells other than macrophages will thus be important.

Third, some supplementary assays might help complete the picture. For example, the fluorescent dyes measured only intracellular levels of reactive molecules in the macrophages. Although these molecules play an important role in driving cellular responses related to oxidative stress, the extracellular levels should also be considered.

Some of these considerations may explain a number of discrepancies between this work and other papers in the literature. For instance, polystyrene nanoparticles and carbon black both generate reactive molecules in a cell-free system, whereas only carbon black generates reactive oxygen species in a macrophage cell line8,9,10.

It is also worth noting that Nel and colleagues prepared the nanoparticles in normal cell culture medium containing fetal calf serum, which contains high levels of antioxidants that may mask the oxidative effects of the nanoparticles. Some studies have exposed cells to nanoparticles in the absence of fetal calf serum to avoid this effect, whereas others have used surfactants to aid particle dispersion. However, little work has been done to determine the impact on toxicity of nanoparticles prepared in these different mediaformulations. It will therefore be essential to compare different dispersion methods to generate a relevant protocol that can be used to standardize assays and aid their interpretation.

Oxidative stress remains the most appealing paradigm for discriminating the adverse effects of different nanoparticles at the cellular and molecular level. The short-term goal for nanotoxicologists is to build on the work of Nel's group, and other similar studies, so that we can relate what we already know about particle toxicology to newly engineered nanoparticles, and also learn more about the mechanisms that are responsible for nanoparticle-induced toxicity. Achieving these goals will promote a safe and profitable nanotechnology.

References

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    Gilmour, P. S. et al. Occup. Environ. Med. 53, 817–822 (1996).

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    Brown, D. M. et al. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L344–L353 (2004).

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    Sayes, C. M. et al. Biomaterials 26, 7587–7595 (2005).

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Affiliations

  1. Centre for Health and Environment, School of Life Sciences, Napier University, Edinburgh, EH10 5DT, UK

    • Vicki Stone
  2. MRC/University of Edinburgh Centre for Inflammation Research, Queen's Medical Research Institute, Edinburgh, EH16 4TJ, UK

    • Ken Donaldson

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https://doi.org/10.1038/nnano.2006.69

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