In the present study, neurotoxicity resulting from exposure to copper nanoparticles was investigated by the morphological study and biochemical assays. Since nanomaterials from the periphery can be retrogradely transported into the nervous system (Praetorius et al. 2007
), it is essential to know the effects of the exposure of neurons to nanoparticles. Sensory neurons innervating the skin are a potential target for such retrograde transport. Copper nanoparticles are now being widely manufactured because of the increasing demand due to their use in several applications. Although the toxicological effects of copper nanoparticles have been shown in vivo
at organ levels in kidney, liver, and spleen (Chen et al. 2006
), our study is the first to report that copper nanoparticles induced toxicity in sensory neurons. The need to characterize nanoparticles before assessing the in vitro
toxicity is crucial. Particle size, morphology, surface area, surface chemistry and particle reactivity in solution are important factors which need to be defined to accurately assess nanoparticles toxicity. The primary sizes of the copper nanoparticles were fairly close to their manufacturer labeled sizes; however, once in neurobasal media, the nanoparticles aggregated to 350–400 nm sizes. Interestingly, all three copper nanoparticles aggregated to nearly the same average size in water and media, with only a minimal increase with increasing particle size. This is interesting in the fact that any differences in the observed effects of the particles would be attributed to other properties of the particles besides the primary particle size. For example, the particles may have different surface features or may behave differently once internalized by the cells. Also, the surface appears to be somewhat oxidized, even after only a few minutes of time in solution, suggesting that the particles are highly reactive. This would need to be verified by surface or elemental analysis of the particles, such as X-ray photoelectron spectroscopy or energy dispersive spectroscopy. However, it has been shown that the surface chemistry and characteristics of these copper nanoparticles began to show changes after one month in solution and they began to exhibit agglomeration in solution after five days and gradually increased their agglomeration by 34 days (Murdock et al. 2008
). In our study, neurons were exposed to copper nanoparticles for only a short duration of 24 h. Since serum appeared to influence agglomeration, we used serum-free defined medium for our cell cultures.
At the end of the 24-h exposure to 40–100 μM concentration of copper nanoparticles, our inverted phase contrast microscopic observation of the DRG neurons showed that some neurons partially detached from the dish and floated in the medium. The detaching of cells from the substratum is an indication of the loss of membrane integrity of the neurons. In addition, many attached neurons showed vacuolation and neurite degeneration. Metal-induced neurotoxicity leading to neurite degeneration has been reported earlier. Methyl mercury has been shown to induce neurite degeneration in primary cultures of mouse dopaminergic mesencephalic cells (Gotz et al. 2002
). Similarly, a 48-h exposure to aluminum chloride resulted in swollen nerve cell bodies in cultured primary cortical neurons from rats along with the presence of beaded and disrupted neurites (Munirathinam et al. 1996
). The membrane compromised, dying neurons would have released the cytoplasmic content to the culture medium. As a result, the LDH values obtained from copper nanoparticles exposed cultures were significantly more compared to the control cultures. Copper nanomaterial appears to be toxic not only to DRG neurons in our study but also to glial cells. A recent report showed dose-dependent (10–100 μM) toxicity on human H4 neuroglioma cells exerted by cupric oxide nanoparticles (Li et al. 2007
). In their study, an automated image analyzer system counted live and dead cells based on the differences in fluorescence between live and dead cells and revealed an increased cell death in H4 neuroglioma cells treated with 10 μM of cupric oxide nanoparticles. However, our results showed that at concentrations of 10 and 20 μM, copper nanoparticles did not exert significant toxicity on DRG neurons. This could be due to differences in the properties of cupric oxide nanoparticles and pure copper nanoparticles and also could be due to the nature of the cells in the two studies and the duration of the nanoparticles exposure. While our cells were fully differentiated, sensory neurons from DRG and exposure duration was 24 h, Li et al. (2007)
used an undifferentiated neuroglioma cell line with an exposure duration of 48 h.
The MTS assay is based on the reduction of the tetrazolium compound to soluble formazan by reductase mostly found in mitochondria, giving a measurement of mitochondrial function in the live cell. The results of the MTS assays showed that exposure to copper nanoparticles in concentrations of 40 μM or higher led to significantly reduced mitochondrial activity or reduced in DRG neurons indicating a reduction in metabolic activity or reduced cell viability. Thus this assay indicates that copper nanoparticles interfere with the reductase activity in mitochondria. This reduction in cell metabolism or viability could have resulted from an increase in oxidative stress. Several reports have shown that transition metals often result in cellular oxidative damage. The accumulation of transition metals in the cytosol may disrupt the intracellular redox status or alter protein conformation and inhibit protein function (Kawata and Suzuki 1983
; Li et al. 1994
). It has been reported that copper inhibits the mitochondrial dehyrdogenases both in vitro
as well as in vivo
and results in the generation of the reactive oxygen species (ROS) (Sheline and Choi 2004
; Valko et al. 2005
). When we stained the copper nanoparticles exposed neurons with rubeanic acid, intraneuronal presence of large deposits of copper was apparent. This copper inside neurons could have inhibited the mitochondrial dehydrogenases and resulted in ROS production. Various studies have shown the resulting cytotoxicity due to the initial induction of lipid peroxidation of the mitochondrial membrane by a metal which might cause decoupling of oxidative phosphorylation, disruption of electron transport, and a decrease in mitochondrial membrane potential (Freedman et al 1989
; Saris and Skulskii 1991
; Mattie and Freedman 2001
). It has been suggested that neurodegenerative process associated with copper overload in Wilson’s disease may be due to the mitochondrial damage, increased production of ROS, and failure of the antioxidant defense mechanisms (Gaetke and Chow 2003
). Also, Pourahmad and O’Brien (2000)
have shown that when isolated hepatocytes are incubated with copper, there is an immediate, rapid increase in ROS production. Thus, in our experiments, decreased neuronal viability following exposure to copper nanoparticles could also be due to an increase in the production of reactive oxygen species. Being a redox-active metal, capable of catalyzing the formation of hydroxyl radicals via a Haber-Weiss or Fenton-like reaction (Cadenas and Davies 2000
; Valko et al. 2005
), copper can also induce oxidative stress by depleting glutathione levels in neurons (Hultberg et al. 1997
). Copper nanoparticles entering the cell therefore could target mitochondria and lead to an increase in oxidative stress.
A three-fold increase in the concentration of copper was seen in the mitoplasts of the SH-SY5Y neuroblastoma cells when exposed to 300 μM of copper sulphate (Arciello et al. 2005
). Our results from rubeanic acid staining show the intracellular deposition of copper nanoparticles at 40–80 μM concentration. Nano-copper appears to enter neurons more readily and at lower concentrations than copper sulphate indicating that copper nanoparticles are capable of easily entering the cell similar to ionic copper.
The results from cell viability (MTS) and cell death (LDH) assays in our study were in accordance with each other. The cultures exposed to copper nanoparticles showed significantly less viability and more death of neurons when compared to those in unexposed control cultures. The higher the concentration and smaller the size, the greater was the neurotoxicity. The toxicity exerted by copper nanoparticles observed in our study was similar to other studies where micro/ionic copper was used (Freedman et al. 1989
; Hultberg et al. 1997
; Mattie and Freedman 2001
; Arciello et al. 2005
). It is interesting to note that when the sizes of particles become small and eventually down to a nanoscale, copper becomes extremely reactive in a simulative intra body environment, and may even lead to changes in gene expression (Meng et al. 2007a
, Wang et al., 2009
In conclusion, our study demonstrated that exposure to copper nanoparticles resulted in significant toxicity to the cultured DRG neurons at concentrations of 40–100 μM but not at 10–20 μM. Although exposure to copper nanoparticles of sizes 40 nm, 60 nm, and 80 nm all had toxic effects on DRG neurons, copper nanoparticles of 40 nm and 60 nm sizes had higher toxic effect than the 80 nm-sized particles. Thus the toxic effect appears to be concentration and size-dependent. The mechanism underlying the toxicological effects observed with the exposure of DRG neurons to copper nanoparticles in the present study can be oxidative stress. Further studies identifying the subcellular location and dynamics of copper nanoparticles are needed to determine the specific mechanism of toxicity in DRG neurons.