Human Toxicology and Effects of Nanoparticles - Environmental and Human Health Impacts of Nanotechnology - page 358

9.2.3 Reactive Oxygen Species and Oxidative Stress

To provide evidence to support the ultrafi ne particle hypothesis, a number of
studies have been conducted to identify a mechanism by which such particles could
be more reactive in biological systems. Such studies have demonstrated that these
low solubility nanoparticles can generate ROS to a greater extent than larger
respirable particles. For example, the plasmid assay has been used to demonstrate
the ability of carbon nanoparticles (Stone et al. , 1998) and a panel of metal nanopar-
ticles (Dick et al. , 2003) to induce ROS production in a cell-free system. The dye
2,7 - dichlorofl uorescin-diacetate (DCFH-DA) has also been used to show that
nanoparticle carbon black (Wilson et al. , 2002) and polystyrene beads (Brown
et al. , 2001) are more potent in a cell free environment, than larger particles at
generating ROS, therefore confi rming the results of the plasmid assay. The same
fl uorescent dye DCFH-DA has also been used to measure nanoparticle induced
intracellular ROS. For example, Wilson et al. (2002) demonstrated that 14 nm carbon
black, but not 260 nm carbon black, stimulated an increase in intracellular ROS in
a macrophage cell line. Due to the propensity of nanoparticles to aggregate in
suspension, Foucaud et al. (2007) suspended carbon black particles in saline supple-
mented with either bovine serum albumin (BSA) (1%) or dipalmitoyl phosphatidyl
choline (DPPC) (0.025%) and investigated the impact of these solutions on particle
aggregation and ROS production. Both BSA and DPPC signifi cantly improved the
stability of the nanoparticle suspension, decreasing the extent of particle agglom-
eration and settling over time. Foucaud et al. (2007) used the DCFH assay to
measure ROS and found that ROS production by 14 nm carbon black was greater
in the DPPC suspension than in either saline alone or the BSA solution. Dispersion
of the 14 nm carbon black particles with either BSA or DPPC also enhanced intra-
cellular ROS detection in the MonoMac6 cell line, suggesting that enhanced disper-
sion also enhances ROS production for the carbon black nanoparticles. Additional
evidence for ROS production by nanoparticles is outlined in Chapter 3.
ROS are of interest because their unpaired electron makes them very reactive,
resulting in damage to many biological molecules including lipids, proteins and
DNA. The body contains a number of antioxidant defence mechanisms to protect
against the damaging effects of ROS, however these defence mechanisms are not
always effective. Oxidative stress is an imbalance between oxidants and antioxi-
dants and can be caused by excessive production of oxidants/ROS or depletion of
antioxidants (Li et al. , 1999; MacNee and Rahman, 2001).
The evidence that ultrafi ne particles can generate ROS in a cell-free environment
suggests that such particles could themselves generate ROS that have the ability
to induce oxidative stress in cells. Furthermore, oxidants can be generated by acti-
vated phagocytic cells such as macrophages (MacNee and Rahman, 2001), contrib-
uting to the pro-oxidant status of a particle exposed organ. In order to identify
whether nanoparticles could induce oxidative stress, Stone et al. (1998) investigated
the effect of 14 nm carbon black on the antioxidant glutathione in an alveolar epi-
thelial cell line. This study identifi ed that 14 nm carbon black induced depletion of
reduced glutathione and accumulation of oxidised glutathione, an effect which was