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Nanometric cerium dioxide was non-irritant in an in vitro EpiDerm test (Park B. et al., 2007, 2008; see Chapter 7.3.1 Skin irritation/corrosion) and was devoid of mutagenicity activity in a bacterial reverse mutation (Ames) test performed in accordance with the OECD 471 test guideline (Park B. et al., 2007, 2008; see Chapter 7.6.1 Genetic toxicity in vitro).

 

Furthermore, nanometric cerium oxide is a member of the list of representative manufactured nanomaterial for phase one of the OECD testing programme (OECD (2008). List of manufactured nanomaterials and list of endpoints for phase one of the OECD testing programme. Series on the safety of manufactures nanomaterials number 6. Environment Directorate - Joint Meeting of the chemicals committee and the working party on chemicals, pesticides and biotechnology - ENV/JM/MONO(2008)13/REV). At the present time, no results of this test programme have been yet released.

 

Several in vitro studies have been performed to study the toxicity on cerium oxide nanomaterials. From these studies, it appears that conflicting results have been observed especially regarding the antioxidant effect of cerium oxide nanomaterials. The preparation method of cerium oxide nanoparticles as well as some cellular physiology related with antioxidant effects among the different types of cells or some media environment could maybe be involved in the differences observed in these studies.

 

Korvsvik and coll. demonstrated that ceria nanoparticles are shown to act as catalysts that mimic superoxide dismutase (SOD) with the catalytic rate constant exceeding that determined for the enzyme SOD (Korvsvik et al., Chem. Commun., 1056 -1058, 2007. Superoxide dismutase mimetic properties exhibited by vacancy engineered ceria nanoparticles).

 

This SOD mimetic activity exhibited by the vacancy engineered ceria nanoparticles was proposed as a mechanism of action for the protective effect of ceria nanoparticles in cells against ROS generated by irradiation. Indeed, Tarnuzzer et al. (2005) studied the ability of engineered cerium oxide nanoparticles to confer radioprotection to human normal breast epithelial cells (CRL8798) and breast carcinoma cells (MCF-7). Cells were treated with nanoceria and irradiated, and cell survival was measured. Treatment of normal cells conferred almost 99% protection from radiation-induced cell death, whereas the same concentration showed almost no protection of tumour cells. It was proposed that nanoceria acts as an antioxidant because of the presence of the mixed valence state of Ce3+ and Ce4+ on the surface, induced by the oxygen vacancies. By changing its oxidation state from Ce3+ to Ce4+, ceria nanoparticles likely scavenge the free radicals generated by irradiation. The autoregenerative antioxidant properties of ceria nanoparticles also appear to be a key component of its radioprotective action.

 

Heckert et al. (Biomaterials 29(18):2705-2709, 2008. The role of cerium redox state in the SOD mimetic activity of nanoceria) studied the role of cerium redox state in the SOD mimetic activity of nanoceria. The results of the study strongly suggest that the surface oxidation state of nanoceria plays an integral role in the SOD mimetic activity of nanoceria and that ability of nanoceria to scavenge the superoxide is directly related to cerium (III) concentrations at the surface of the particle.

 

Furthermore, Schubert and coll. (2006) have demonstrated that Ceria nanoparticles decreased the endogenous ROS induced by glutamate and showed cell protective effects in cultures of hippocampal nerve (HT22) cell line which are derived from the rodent nervous. In this study, the authors claimed that nanoceria act as a free radical scavenger.

 

Das et al. (2007) also showed that ceria nanoparticles offer neuroprotection to adult rat spinal cord neurons. Spinal cord neurons and other CNS neurons are prone to be damaged due to oxidative stress, both in vitro and in vivo. However, when cerium nanoparticles were added to the cultures with hydrogen peroxides, a significantly higher number of surviving cells were shown as compared to the non-treated control group.

The authors suggested that the presence of the mixed valence states of Ce3+ and Ce4+ on the surface of the nanoceria act as an anti-oxidant that allow the nanoparticles to scavenge free radicals from the culture system. The auto-regenerative anti-oxidant property of these nanoparticles appears to be the key to its neuroprotective action.

It was also observed that CeO2 nanoparticles protect against the progression of cardiac dysfunction and remodelling by attenuation of myocardial oxidative stress (Niu et al., 2007). In this study, the authors observed that treatment (intravenous injection) with CeO2 nanoparticles in a transgenic mouse (MCP mice) model with cardiac-specific expression of MCP-1 inhibited monocyte/macrophage infiltration and suppressed the production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1) in the myocardium. CeO2 nanoparticles also inhibited peroxynitrite formation and levels of NOx, and suppressed the induction of endoplasmic reticulum stress-associated genes in the heart. Thus, the authors concluded that CeO2 nanoparticles likely suppress myocardial inflammatory process and oxidative and endoplasmic reticulum stress and thereby inhibit the progression of left ventricular dysfunction and remodelling in the transgenic murine model of ischemic cardiomyopathy.

 

Furthermore, cerium oxide nanoparticles caused very little inflammatory response but not cell loss in human aortic endothelial (HAECs) cells exposed for 4 hours at concentrations up to 50 µg/mL, as measured by the mRNA expression of the inflammatory markers ICAM-1, IL-8 and MCP-1 (Gojova et al., 2009).

 

Xia et al. (2008) observed that CeO2 exhibited no cytotoxic effects in BEAS-2B (transformed human bronchial epithelial) cell and RAW 264.7 (phagocytic) cells. Moreover, CeO2 protected both cell types tested against a secondary oxidative stress stimulus, suggesting that the electronic properties of CeO2 may lead to antioxidant protective effects.

 

In contrast, Lin et al. (2006) showed that in the human bronchoalveolar (A549) carcinoma-derived cells treated with 20 nm CeO2 nanoparticles, reactive oxygen species (ROS) were induced and cell viability was decreased. The elevated oxidative stress was found to increase the production of MDA and LDH, indicators of lipid peroxidation and membrane damage, respectively.

 

While exposure of human dermal fibroblasts to cerium oxide nanoparticles resulted in no cytotoxicity, DNA damage was detected in fibroblasts using a Comet assay (Auffan et al., 2009). The authors suggested that this may be related to oxidative stress induced by reactive oxygen species generation. Redox processes between cerium oxide nanoparticles and molecules present in cell nutritive medium (mainly proteins) were responsible for 8 ± 2% reduction in the Ce3+ oxidation state.

 

Furthermore, Park E-J, Choi J, Park Y-K and Park K (2008) observed that exposure of the cultured BEAS-2B (transformed human bronchial epithelial) cells to ceria nanoparticles led to cell death, ROS increase, GSH decrease, and the inductions of oxidative stress-related genes such as heme oxygenase (HO)-1, catalase, glutathione S-transferase, and thioredoxin reductase. The increased ROS by cerium oxide nanoparticles triggered the activation of cytosolic caspase-3 and chromatin condensation, which means that cerium oxide nanoparticles may exert cytotoxicity by an apoptotic process. In another study performed by the same group (Eom H-J and Choi J, 2009), it was suggested that CeO2 nanoparticles provoke oxidative stress in BEAS-2B cells and, in response to this, mainly the p38 MAP kinase signalling pathway seems to be activated. Induction of HO-1 can be interpreted as a cellular defence mechanism against oxidative stress, despite the absence of CeO2-induced NF-kappa B activation.

 

In contrast, Park E-J et al. (2008) observed no cytotoxic effect of nanoceria in human brain fibroblast (T98G) and rat cardiomyocytes (H9C2) cells after a 98-hour exposure.

 

Furthermore, cytotoxicity was not observed either following a 24-h exposure of hepatocytes (C3A cell line or primary trout hepatocytes) to a suspension of nanoparticular cerium oxide (Gaiser et al., 2009) or after a 3-h exposure to an aerosol of nanoparticulate cerium oxide of organotypic cultures of Wistar rat lung slices (Fall et al., 2007).

 

Cerium oxide nanoparticles was also found non-cytotoxic in an BS EN ISO10993-5guideline compliant test when applied as a 1 cm² area on agar overlay (Park Bet al, 2007, 2008).

 

Interestingly, it was observed in some of these studies that effects of nanoceria were independent of the particle size (Schubert et al, 2006, Eom H-J and Choi J, 2009).

 

Conclusion

From the results observed with cerium oxide nanomaterials in in vitro skin irritation test and in bacterial reverse mutation (Ames) as well as in ecotoxicological studies, no obvious differences with results observed with micrometric (bulk) cerium oxide were evidenced. However, since cerium oxide nanomaterial is currently under OCDE testing programme (OECD (2008). List of manufactured nanomaterials and list of endpoints for phase one of the OECD testing programme. Series on the safety of manufactures nanomaterials number 6. Environment Directorate - Joint Meeting of the chemicals committee and the working party on chemicals, pesticides and biotechnology - ENV/JM/ MONO(2008)13/REV), the present registration dossier will be updated at the release of the results of the OCDE studies.