During the last few years, research on toxicologically relevant properties of manufactured nanoparticles has increased at an exponential rate. Currently, most of the toxicological work on nanoparticles have been generated with a small set of nanoparticles, such as carbon black, C60, TiO2, iron oxides and amorphous silica, which have been manufactured by the chemical industry for some decades and are produced in bulk quantities each year [5, 37]. There is evidence that a number of factors are likely to contribute to the toxicity of nanoparticles, including particle number and size, surface area and charges, and chemical composition . Nevertheless, experimental conditions, type or dose of nanoparticles used, or the nature of the assays can also modulate the assessment outcome. It is, therefore, necessary to establish an efficient system to determine the genotoxic events induced by nanoparticles both in vivo and in vitro.
Genetic alterations, such as point mutations, chromosomal rearrangements, recombination, and insertions or deletions of genes, are thought to be one of the earliest cellular responses caused by physical and chemical carcinogens and may play an important role in the initiation and progression of carcinogenesis . Previous studies from this laboratory have shown that the gpt delta transgenic mouse system provides a unique opportunity to assess the mutagenic potential of asbestos fibers . The gpt mice carry tandem repeats of λ G10 DNA in the chromosome, which are retrievable as phage particles by an in vitro packaging reaction. The rescued phages are then used to quantify the mutation yield upon exposure to genotoxic agents. The Spi- selection based on deletions extending into or through both the redBA and gam genes is an efficient mutation assay system for detecting small to kilo-base-sized deletions in different cells, organs, and tissues . Since gene mutation, mitotic recombination, chromosome loss, and interstitial deletion largely contribute to the development of malignancy, the establishment of the gpt delta transgenic mouse mutation model may provide new insight on understanding nanoparticle-induced mutagenesis. Our present findings demonstrated that TiO2 at nano-scale increased the mutant yield at the gam and redBA loci in MEF cells, while TiO2 at micro-scale had little effect on the mutation induction. These data were consistent with several in vivo and in vitro findings that, upon transition from the micro-scale to nano-scale size range, diameter of inhaled or instilled particles are important factors influencing the toxicity response [19, 40, 41]. The BET surface area for TiO2 5 nm was increased by 3-fold from 38.2268 m2/g to 114.1261 m2/g as compared to TiO2 40 nm, however, there was no statistically significant difference among groups expsoed to either TiO2 5 nm or TiO2 40 nm at the same dose (Figure 2), which are in conflict with the notion that toxic response is generally considered to be higher in particles with large surface area than those with smaller area . Although a surface area dependence and correlation have been observed in instillation studies , recent evidence from rats and mice showed that the surface area for TiO2 nanoparticles was not a significant factor in inflammatory response [12, 43]. In addition, we showed here that C60 was cytotoxic and mutagenic in transgenic MEF cells, although the exact mechanisms are largely unknown.
Endocytosis is a conserved process in eukaryotes by which extracellular components are taken up into cells by invagination of the plasma membrane to form vesicles that enclose these materials . There are several possible uptake pathways for internalizing nanoparticles, such as phagocytosis, macropinocytosis, clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin-caveolae-independent endocytosis (5, 45). Several recent evidence has shown that certain nanoparticles, such as iron oxide and silica, as well as carbon nanotubes, are internalized in cells via the endocytic pathway [46, 47]. After 24 h incubation, we observed that the cellular granularity of MEF cells exposed to TiO2 particles was increased in a dose-dependant manner. In contrast, C60 had no effect on the cellular granularity, which might be due to their low contrast and small diameters. Our results with the lipid raft-disrupting agent Nystatin, which binds to cholesterol in cell membranes and disrupts the formation and trafficking of caveolae, provided further support of the idea that the endocytotic process modulated the mutagenic response of nanoparticle treatment . Given C60 is lipophilic, it is possible that C60 may interact with plasma membrane lipids and exert toxicity directly in the absence of cellular uptake . It is also likely that C60 interact with cell membrane receptors to trigger or alter intracellular signal transduction pathways. Due to high energetic adhesive forces close to the surface, nanoparticles are easily agglomerated to form larger particles. Thus, whether single particles or agglomerates are important in the genotoxicity of nanoparticles has not been identified yet.
The mechanism of oxidative stress induced by nanoparticles is not well understood. There is evidence that free radicals can be induced at the surface of nanoparticles such as single-wall carbon nanotube (SWCNT), semiconductor quantum dots, TiO2, environmental particles (e.g. PM-10), asbestos, and a range of man-made fibers [14, 48, 49]. Among the most biologically active oxyradicals such as superoxide anions (O2
.), hydrogen peroxide (H2O2), and hydroxyl radical (OH·), NO is relatively long lived and catalyzed by nitric oxide synthase (NOS) . The few cell culture experiments on nanoparticles, such as metal oxides and quantum dots, have identified particles within or around the mitochondria [17, 33]. Since mitochondria constitute a major locus for the intracellular formation and reactions with NO, it is likely that multiple radical species are involved in the genotoxic response of TiO2 nanoparticle and C60 exposure. NO reacts with O2
-. and can be rapidly converted into more reactive nitrogen compounds such as ONOO- that can cause nitration of proteins, hydroxylation or nitration of DNA, and mutations  Nano-sized TiO2 exposure has been reported to increase the production of NO and oxidative DNA damage in human bronchial epithelial cells . In the present study, TiO2 nanoparticle exposure dramatically increased the generation of ONOO- in MEF cells. It should be noted that nano-TiO2 particles in the anatase crystal phase were reported to be superior catalysts and more cytotoxic as compared to the rutile particle type, which might be due to differences inherent in the crystal structures of the two phases, rather than differences in surface area (11). There is evidence that the unique structure of C60 facilitates absorption of light and transfer of this energy to triplet oxygen, thereby forming the highly reactive singlet oxygen state, which may cause oxidative damage in exposed organisms . Recent reports have showed that C60 induces cytotoxic effects via the induction of reactive oxygen species in mouse cells, human cells, and fish. However, it should be noted that some data indirectly suggest that oxyradical-mediated cytoxicity of C60 might not be an inherent property of pure C60, but rather a result of the residual presence of tetrahydrofuran (THF), the organic solvent used for C60 preparation, which remains intercalated into its lattice . Here, C60 suspension prepared by long-term stirring in water. The oxidation of DHR 123 by ONOO-, as detected using confocal microscopy, provided direct evidence that C60 induced a dose-dependent increase of ONOO- in single cells, which could be inhibited by the NOS inhibitor L-NMMA. Moreover, the mutation yields induced by either nano-sized TiO2 or C60 in MEF cells decreased by concurrent treatment with L-NMMA, indicating a key role of ONOO- in the mechanisms of nano-sized TiO2 and C60-induced genotoxicity. It's woth notice that the redox events might be caused by the signaling events associated with the transporting of naoparticles into the cellular structure, rather than the chemical composition/surface area combiantion of the nanoparticles.
COX-2 is a member of the COX family, which plays important roles in modulating cellular inflammation, carcinogenesis and genomic instability . Nitric oxide synthase, which is critical to the biosynthesis of ONOO-, has been shown to be involved in the regulation of COX-2 expression [36, 54]. Since COX-2 is the initial and rate-limiting enzymatic step in the metabolism of arachidonic acid into a complex group of signaling lipid mediators, the particle-induced oxidative stress may lead to transmit external signals into the cell and activate COX-2 signal pathway. In the presence of NS-398, a specific inhibitor of COX-2 , the genotoxic effects of both nano-sized TiO2 and C60 was reduced dramatically in MEF cells, thereby establishing the functional link for the role of ONOO- and COX-2 in mediating the genotoxic events of both nano-sized TiO2 and C60.
The toxicological data specific to nanoparticles remains insufficient currently [5, 56]. However, the potential toxicity of nanoparticles has attracted attention because of their apparent similarities to asbestos and other carcinogenic fibres/particles. Our present studies provided direct evidence on the genotoxicity of two specific types of manufactured nanoparticles, TiO2 and C60, and highlight several key health risk assessment issues associated with manufactured nanomaterial, such as the paucity of information on nanoparticle toxicology and exposure assessments as well as the extent to which nanoparticle toxicity can be extrapolated from existing particle and fiber toxicology databases.