Nanoparticulate Vanadium Oxide Potentiated Vanadium Toxicity in

Occupational Exposure Limits (SCOEL), and German Sen- atskommission zur ..... account, the chance and speed of oxidation from V2O3 to. V2O5 increases ...
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Environ. Sci. Technol. 2007, 41, 331-336

Nanoparticulate Vanadium Oxide Potentiated Vanadium Toxicity in Human Lung Cells JO ¨ RG M. WO ¨ RLE-KNIRSCH,† KATRIN KERN,† CARSTEN SCHLEH,† CHRISTEL ADELHELM,‡ CLAUS FELDMANN,§ AND H A R A L D F . K R U G * ,† Department of Molecular Environmental Toxicology, Institute of Toxicology and Genetics,Forschungszentrum Karlsruhe, Institut fu ¨ r Materialforschung I, Forschungszentrum Karlsruhe, Institut fu ¨ r Anorganische Chemie der Universita¨t Karlsruhe, D-76131 Karlsruhe

Metal oxides may hold, as nanosized particles, a toxic potential to human health and the environment that is not present in the bulk material. Due to the high surface-tovolume ratio, small amounts can lead to strong oxidative damage within biological systems, impairing cellular functions as a consequence of their high surface reactivity. We report here on a new nanosized V2O3 material that has a needle-like structure with diameters of less than 30 nm and variable lengths. The potentiated toxicity of nanoscale vanadium oxide (V2O3) compared to bulk material is demonstrated here in human endo- and epithelial lung cells, and might be due to the higher catalytic surface of the particles. Reduction in cell viability is almost ten times stronger and starts with lowest concentrations of “nanoscaled” material (10 µg/mL). Vanadium oxide leads to an induction of heme oxygenase 1 (HO-1) in a dose dependent manner in ECV304 cells whereas a reduction in protein levels can be observed for the epithelial cells (A549). Lipid peroxidation can be observed also for “nanoscaled” vanadium oxide to a much stronger extent in macrophages (RAW cells) than for bulk material. The observed effects can not only be explained by oxidation from V2O3 to V2O5 as there are significant differences between the novel nano vanadium and all used bulk materials (V2O3 and V2O5). It appears rather to be a nanoeffect of a high surface reactivity, here coupled with a yet unknown toxicity potentiating effect of a technically important catalyst.

Introduction Nanotechnology is a fast, exciting, and promising new technology with rapidly growing knowledge. Nanomaterials are on the same scale as most elements of living cells, including proteins, nucleic acids, lipids, and even cellular organelles. V2O3 is discussed with concern to the following areas of application: lithium ion batteries, field effect * Corresponding author phone: + 49 7247 82 3262; fax: + 49 7247 82 3557; e-mail: [email protected]. † Institute of Toxicology and Genetics, Forschungszentrum Karlsruhe. ‡ Institut fu ¨ r Materialforschung I, Forschungszentrum Karlsruhe. § Institut fu ¨ r Anorganische Chemie der Universita¨t Karlsruhe. 10.1021/es061140x CCC: $37.00 Published on Web 12/06/2006

 2007 American Chemical Society

transistors, thermochromic materials, redox catalysts, and infrared switching devices. In connection to nanoparticles it must be asked how man-made nanostructures can possibly interact or influence biological systems? On the one hand, nanosystems are specifically engineered to interact with biological systems for particular medical or biological applications (1-5). On the other hand, the large scale production of nanoparticles for either non-medical applications or as side products of combustion processes may have an effect on organisms in all parts of our environment. Since the 1970s, investigations are continually increasing that analyze the use of nanoscale structures, e.g., synthetic liposomes, for drug transport and comparable applications. But nanostructured materials come into contact with biological systems not only by their use for drug delivery systems or gene transfer. They are also produced for food and cosmetic chemistry and many other technical applications (6). Metal oxides, for example, are used in a broad range of applications in nearly all fields of technology and industry. As additives in sun screens and textiles ,they banish UV light, they clear apple juice and beer, and they help to degrade toxic chemical waste in a very efficient manner (7, 8). Data on toxicity are available, especially for those materials that have been used for decades (TiO2, SiO2, carbon black), but very little is known on the biological action and interaction with cellular structures for most newly developed nanomaterials. It is assumed that nanoparticles in sintered or agglomerated form (to larger structures) lose most of their vast toxic potential to human health, which appears in primary particles or small aggregates. Therefore, assessment of nanoscaled metal oxide toxicity is focused on free and primary particles. Recently, for vanadium pentoxide, major changes in perception as an occupational toxicant and MAK values have been revised (9). This was done with participation of the U.S. American Threshold Limit Values Committee (TLV), European Scientific Committee on Occupational Exposure Limits (SCOEL), and German Senatskommission zur Pru ¨ fung Gesundheitsscha¨dlicher Arbeitsstoffe der Deutschen Forschungsgemeinschaft (DFG) regulatory commissions (9). Vanadium pentoxide occupational exposure limits have then been abandoned for 2005, and since then, the latest changes are discussed in all of these committees. In this work, we investigated the potentiated toxicity of a novel vanadium oxide catalyst in nano form by analyzing various biological endpoints, respectively.

Materials and Methods Cell Culturing. Human endothelial cells derived from an umbilical cord were obtained as a cell line (ECV304) from the European Collection of Cell Cultures. The human alveolar epithelial cell line A549 (ATCC, CCL-185) (10, 11) and mouse alveolar macrophages RAW 264.7 (12) were obtained from American Type Culture Collection (Rockville, MD). Human A549 cells and mouse RAW 264.7 were grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Karlsruhe), ECV304 cells were grown in M199 (Invitrogen, Karlsruhe), all supplemented with 10% heat inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 µg/mL penicillin, and 100 U/mL streptomycin. The cells were grown in a humidified incubator at 37 °C (95% room air, 5% CO2). Nanoparticles. Based on a polyol-mediated synthesis, a manifold of nanoscale oxides is accessible. According to this type of synthesis, nanoscale oxides are precipitated in a highboiling, multivalent alcohol (so-called polyol), such as glycerin or diethylene glycol. Due to its coordinating properties, the alcohol is used on one hand to limit particle growth, and on VOL. 41, NO. 1, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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the other, to avoid aggregation (13, 14). A typical recipe for the polyol-mediated synthesis rod-shaped V2O3 is as follows: 3.50 g VO(i-OC3H7)3 (ABCR, 99%) were added to a mixture of 25 mL diethylene glycol and 25 mL water. In addition, 400 mg NaOH (Aldrich, >97%) were dissolved in 50 mL diethylene glycol (Merck, 99%) and heated to 180 °C. At this temperature, the vanadium containing solution was added dropwise in 1 h under vigorous stirring. Water and alcohol were continuously distilled off. The yellow to orange color of the vanadium containing solution turned deep green right after addition to the alkaline solution. The characteristic green color indicates an immediate reduction of V5+ to V3+. Herein, diethylene glycol acts as the reducing agent (15). When the solvent addition was finished, the temperature was kept at 180 °C for an additional hour. As a result, a colloidal stable, dark green suspension containing needleshaped particles, about 500 nm in length and 25 nm in diameter was realized. By centrifugation the solid material was removed from the liquid. Resuspension in ethanol, followed by centrifugation, which was carried out to remove diethylene glycol completely, yielded a dark green amorphous powder. V2O3 nanoparticles were stored in DEG and, prior to use, sedimented by centrifugation. Excess of DEG was discarded and particles diluted to appropriate concentrations with culture media. Before experimental use, nanoparticle solution was sonified for 10 times 1 s. at 70 W. Bulk material (CAS nos. 1314-34-7 and 1314-62-1) was purchased from SIGMA and treated equally. Determination of Vanadium Oxide Solubility. Vanadium oxide bulks as well as the nanomaterial were prepared as described for the experiments, but in a concentration of 1 mg/mL. Then the chemicals were incubated in bi-distilled water and kept at identical conditions for a time period of up to 48 h (5% CO2, 37 °C, darkness) and samples were taken for analysis at indicated time points (as quadruplicates). After centrifugation (21 000 g, 30 min, 4 °C) part of the above standing liquid has been withdrawn by pipet, diluted with deionized water, and acidified by subboiled nitric acid. The vanadium content has been analyzed by ICP-OES (PerkinElmer: OPTIMA 4300 DV) in comparison with matrix matched calibration solutions covering the concentration range of the samples. Three emission lines of vanadium have been used: 311.071, 309.310, and 292.402 nm. The detection limit calculated by 6 times of the standard deviation of 30 acid blanks measured between the samples varies insignificantly between 0.006 and 0.007 mg/L. Figure S2 shows typical emission spectra of the solutions from the solubility experiments after 6 h. Figure S3 displays the vanadium solubility over time as comparison of bulk to nano materials. MTT-Viability Assay. Cells were grown in 96 well plates (NUNC, Germany) overnight. Each cell line was seeded at 2.5 × 104 cells into the wells of a 96-well plate in triplicate. Cells were treated with the described particle suspensions in concentrations of 10, 25, 50, 75, and 100 µg/mL in complete culture medium for 48 h. Cytotoxicity was determined by measuring the reduction of the yellowish water soluble MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, SIGMA) to water insoluble formazan. Formazan extraction was done with isopropanol/HCl and measured at 550 nm in a multiplate reader against isopropanol/HCl. The results are given as relative values to the negative control in percent, whereas untreated (negative) control is set to be 100% viable. Western Blot Analysis. Cells/well (5 × 105) were seeded in six well plates. After 24 h, cells were incubated with particles. Cells were harvested and immediately homogenized with ice-cold NP-40 lysis buffer [150 mM NaCl, 50 mM Tris (pH 8), 5 mM EDTA (pH 8), 1% NP-40, 3% PMSF, 0,2% Aprotinin, 0,2% Leupeptin] on ice for 20 min. The cells were centrifuged at 16 000 g at 4 °C for 15 min. The supernatants 332

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were decanted and stored at -20 °C. The protein concentration was determined via Bradford assay. Ten micrograms of protein were loaded on each lane of a 12.5% polyacrylamide gel. The proteins were separated under denaturing conditions (1% SDS) for 1 h at 170 V. The proteins were transferred to a PVDF (ECL) or Immobilion FL membrane (Odyssey, LI-COR Biosciences) for 1 h at 100V. The membranes were blocked at 4 °C for 2 h with 10% milk in PBS- 0.03% Tween or Odyssey blocking buffer. The blots were incubated with the first antibody over night [ECL: HO-1 1:1000; PCNA 1:2,000; actin 1:2,000 (all antibodies: Santa Cruz Biotechnology, Heidelberg, Germany); for use in the Odyssey system: all first antibodies 1:5000]. After washing in PBSTween the blots were incubated with the second antibody for 2 h [anti rabbit 1:1000 (Amersham, Freiburg, Germany); anti mouse 1:2000 (Dako, Hamburg, Germany), anti goat 1:4000 (Rockland, Biomol, Hamburg, Germany), Odyssey: 1: 20 000 (Rockland, Biomol, Hamburg, Germany)]. The signals were detected by ECL method or Odyssey reader (Licor Bioscience, Bad Homburg, Germany). DCF-Assay. Cells (25 000) were seeded in 96 well plates. After 48 h the cells were washed in Hank’s balanced salt solution (HBSS) then incubated with 2′‚7′-dichlorodihydrofluorescein (50 µM) (Molecular Probes Eugene, OR) in HBSS for 40 min at 37 °C. The cells were washed twice with HBSS (Gibco, Karlsruhe, Germany), incubated with particles, and immediately measured in the fluorescence reader (MWGBiotech AG, Ebersberg, Germany) at 485 nm excitation and 530 nm emission wavelengths and again measured 1 h later. The stimulator of NO release Sin-1 (3-morpholinosydnonimine, hydrochloride, Molecular Probes Eugene, OR) was used as a positive control at 10 µM final concentration. Lipid Peroxidation. Five million RAW 264.7 cells were grown in a 3 mL culture media in a six-well plate (NUNC, Germany) overnight. Samples were incubated with indicated particle concentrations for 24 h. TBARS assay (thiobarbituric acid) was performed after Jentzsch (16) with modifications. Cells were lysed with 1 mL 2% SDS, mixed with 1 mL 0.2 M phosphoric acid and 125 µL 30 mM BHT (di-tertio-butylhydroxy-toluene). Thiobarbituric acid was added to 0.11 mol/ L, samples were shaken and incubated for 60 min in a water bath at 90 °C. Reaction was stopped on ice, and TBA complex was extracted with 800 µL n-butanole and 80 µL of a saturated NaCl solution. After mixing and centrifugation of the samples, photometric measurement in a 96-well plate (Nunclon, Germany) was performed with the supernatant at 535 and 572 nm. MDA standards and equivalents were calculated from standard preparation as described elsewhere (16). EM-Preparations. Particles were prepared as for use in the experiment and spotted on 75-mesh Formvar coated copper grids, dried, and analyzed with a Zeiss 109T transmission electron microscope (Oberkochen) or a Zeiss Supra 55VP scanning electron microscope.

Results Electron micrographs of this new vanadium oxide (V2O3) demonstrate a needle-like structure for this “nanoscaled” material (Figure 1). These needles have an average diameter of 25 nm, and the length varies between 100 and 1000 nm. Vanadium oxide needles look like vanadium nanotubes (described elsewhere, refs, 17, 18) and are found in bundles or as solitary fibers after preparation. BET analysis (BET: Brunauer-EmmettTeller) evidenced a specific surface of 74.9 m2g-1 for this nano material. Solubility was highest for nanoscaled V2O3, followed by bulk V2O5 and V2O3, as can be seen in Figure S3 in the Supporting Information. At first, we determined cell viability after treatment of human lung epithelial cells (A549) with various concentrations of vanadium oxides in its different forms. The influence

FIGURE 1. Electron micrographs of nanoscaled vanadium oxide. SEM (scanning electron micrograph) image (A) taken right after preparation of nano V2O3 in DEG (diethylenglycol) and the same material after aqueous preparation for (B) TEM (transmission electron micrograph). SEM image displays material as hedgehog like clusters, whereas TEM shows needle like vanadium oxide particles marginally clustered and solitaire. The decrease in viability could be observed at concentrations as low as 10 µg/mL with nanoscaled material, whereas for the same concentration of the bulk material, almost no loss in viability could be detected. Dose-dependent decrease in viability continues for nanoscaled vanadium oxide steep and reaches a final level of about 3% at maximum concentrations used within these experiments (100 µg/mL). At the same high concentration, the reduction in cell viability induced by the V2O3 bulk material is as low as 27% (leaving 73% viable behind). V2O3 can be slowly oxidized to V2O5 in aqueous solutions, thereby becoming much more water soluble. Thus, we also compared the nanoscaled vanadium trioxide with V2O5 bulk material (Figure 2B). Again, only a slight decrease in viability can be observed for the bulk material at lower concentrations ranging from 10 to 25 µg/ mL. Overall, a strong dose-dependency was found, with a final viability for V2O5 bulk material of about 17% at 100 µg/mL; this value is still significantly (p < 0.05) different from that of V203 nanomaterial at equal concentrations. Similar results have been obtained with ECV304 human endothelial cells also with MTT and LDH assay (data not shown), although these cells are not as sensitive to vanadium oxides as the A549 cells. Oxidative stress and the formation of reactive oxygen species (ROS) were shown to be one of the key mechanisms in cellular defense after particle uptake (19). Incubation of A549 cells with “nanoscaled” V2O3 revealed a strong increase in overall ROS production (Figure 3) up to nearly the 4-fold values over untreated control cells. FIGURE 2. MTT-viability assay for A549 cells with V2O3. Human A549 cells were exposed to nanoscaled vanadium oxide (V2O3; filled circles A + B) as well as to bulk material (open circles: V2O3:A; V2O5:B) for 48 h in complete media. The MTT assay measures the activity of mitochondrial dehydrogenases in living cells. Values are the mean of >4 experiments ((SD) measured in quadruplicates. on cell viability and ROS induction of remaining DEG was tested with a 100-fold excess; without negative effects (data not shown), therefore, DEG was considered to be nontoxic in the concentrations used. Most important was the comparison of nanoscaled vanadium oxide with its complementary bulk material. The results demonstrate a strong decrease in cell viability after incubation with amounts of nano vanadium oxide as low as 10 µg/mL (Figure 2). It is clearly visible that cell viability is reduced in A549 cells significantly more after incubation with the nanoscaled vanadium oxide (V2O3) than after treatment with the bulk material (Figure 2A).

The increase in reactive oxygen species for both bulk materials could be observed as well, and to an even greater extent, respectively. ROS levels are significantly higher for bulk materials at concentrations above 75 µg/mL comparing nano and bulk material. Treatment of cells with the overall very toxic vanadium pentoxide bulk material induces the highest ROS levels and reaches 7-fold amounts compared to control in both cell types (A549 data not shown). Bulk V2O3 increases ROS levels sigificantly over “nanoscaled” V2O3 only at highest concentrations. Thus, the induction of ROS in human lung endo- and epithelial cells by the “nanoscaled” V2O3 might be explained by rapid conversion to V2O5 in aqueous solution. At concentrations of about 75 µg/mL, only very few cells are viable in the nanomaterial treatment; therefore, it seems reasonable that ROS production is lower than for any observed bulk material. Heme oxygenase 1 (HO-1) has been found to be important within the regulation of oxidative stress as well as other physiological processes (for review see Wagener, ref, 20). A VOL. 41, NO. 1, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Determination of ROS by DCF-assay in ECV304 cells. ECV304 cells were treated with different vanadium oxides in a concentration range from 10 to 100 µg/mL for 48 h. Values are the mean of more than three experiments ((SD). Nanoscaled vanadium oxide (fine hatched bars) is compared to bulk V2O3 (coarse hatched bars) and bulk V2O5 (cross hatched bars). Significance calculated by student’s t-test (*) p < 0.05; (**) p < 0.01.

FIGURE 4. Western blot analysis of HO-1 (heme oxygenase 1) protein expression levels. Upper lane: time course of HO-1 protein levels after vanadium oxide treatment. Protein levels for HO-1 were detected in ECV304 and A549 cells after treatment with 21 µg/cm2 of nano scale vanadium oxide. Lower lane: concentration dependency in ECV304 and A549 cells treated with nano scale vanadium oxide in a range from 6 to 62 µg/cm2 for 24 h. change in induction can be seen in ECV304 cells at concentrations between 31 and 62 µg/cm2 (Figure 4). A heme oxygenase-1 specific antibody was used to detect protein levels in cell lysates. Equal amounts of protein were loaded as confirmed by co-staining with actin or PCNA specific antibodies. As one can see in Figure 4, both cell types differ markedly in their basal expression of HO-1. Whereas the human epithelial cell line A549 has a very high basal HO-1 protein level; the HO-1 is not detectable within the endothelial cell line ECV304. On application of “nanoscaled” vanadium oxide, a strong induction can be seen in ECV304 cells (Figure 4) and as described elsewhere (21) a reduction of HO-1 protein content in A549 (Figure 4) in a dose dependent manner. Both effects are dose- and time-dependent processes, even if they go in opposite ways. Protein expression starts in ECV304 cells between 31 and 46 µg/cm2, whereas HO-1 334

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FIGURE 5. Comparison of lipid peroxidation levels in mouse macrophages after treatment with different vanadium oxides. Mouse RAW 264.7 macrophages have been incubated for 24 h and lipid peroxidation was determined from whole cell lysates. Nanoscaled vanadium oxide (fine hatched bars) is compared to bulk V2O3 (coarse hatched bars) and bulk V2O5 (cross hatched bars). Significance calculated by student’s t-test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001 (n g 4). disappears in A549 cells at comparable concentration levels. Up regulation of protein expression is obvious between 6 and 12 h of treatment, while degradation takes 1-2 days. These reactions cannot be observed for the bulk material (Figure S1, Supporting Information) of V2O3 in either cell line. Only cells treated with either nanoscale V2O3 or bulk V2O5 react as described above. Besides protein synthesis and alterations in protein levels of cells, lipid metabolism and peroxidation is a sensitive parameter for toxic effects of various environmental pollutants with oxidative properties (22-25). Within macrophages (RAW 264.7) the production of malondialdehyde (MDA) during lipid peroxidation could be measured photometrically as adduct with thiobarbituric acid. It has been described for fullerenes (24) that lipid peroxidation can take place upon nanomaterial incubation, resulting in cell death (25). After incubation of macrophage-like cells with vanadium oxides of different sizes, lipid peroxidation is much stronger for nanoscale V2O3 than with the respective bulk material (Figure 5). The peroxidation of lipids is induced at low concentrations of nanoscaled V2O3 and a comparable effect can only be seen with the known toxic bulk material V2O5 (Figure 5), whereas the V2O3 bulk material has nearly no effect during a 24 h treatment. The oxidative stress indicated by the increase in DCF-fluorescence that has been described and observed in human endo- and epithelial lung cells (Figure 3) has been confirmed in this study with RAW 264.7 mouse macrophages, another cell type from a different species, suggesting a species independent induction of ROS and heme oxygenase-1 for vanadium oxide (Figure 4 and 5). Lipid oxidation differs significantly if cells were treated with nano vanadium oxide or the same compounds as conventional bulk material (Figure 5). Peroxidation can be observed with all treatments, but is weaker for V2O3 bulk and does not continue to increase, whereas this can be observed for “nanoscaled“ V2O3 or V2O5 bulk. With a treatment of any of the mentioned materials, the increase can be as high as 600% over control (Figure 5). Interestingly, a significant difference (p < 0.001) can be observed already at a concentration of 25 µg/mL in macrophages between the two bulk materials.

Discussion It has been described recently that ultrafine (20 nm) preparations of titania (TiO2) cause a significant loss in

viability, whereas fine (220 nm) particles do not have such an effect (26). The same correlation for size-effects has also been described for ultrafine and fine nickel and cobalt (27, 28). Additionally, an increase in fibrogenic mediators, like procollagen, can be observed, that appears to be stronger for ultrafine preparations (29) than bulk. A similar observation was made here. Proliferation of macrophages is impaired in these samples as well, and to a greater extent than in fineparticle-treated controls (26). The increased solubility and accessibility of vanadium from nanoparticles to the cells enhances the described toxic effects. The soluble form of nano vanadium induces peroxidative effects stronger than comparable bulk materials, which has been demonstrated in DCF and lipid peroxidation measurements. Inhalation studies revealed a higher pulmonary deposition in rats with ultrafine CdO (40 nm) aerosol than was measured with fine CdO particles (30). Bermudez and his colleagues suggested that retardation of particle clearance in mice and rats is based on pulmonary particle overload that has been achieved in these animals by treatment with high doses of nanoscale titania (31). V2O3 bulk material is known to be poorly soluble in water. Thus, it is reasonable why it has a higher LD50 than watersoluble V2O5. Comparison of LD50 values for V2O3 and V2O5 bulk material exhibit a great difference: the acute toxic oral dose for V2O3 in rats is 566 mg/kg and for V2O5 as low as 10 mg/kg. The values for “nanoscaled” V2O3 are even below the ones for V2O5 (in our experiments) but cannot be compared in absolute matter because of differences in the assays used. LD50 values calculated for our exposure system in the lung are 5 times lower than those for any bulk material investigated so far (LD50 nano V2O3 16 µg/mL, LD50 bulk V2O3 higher than 100 µg/mL in human lung epithelial cells). The high reactivity of this material can be explained by its high surface to volume ratio. Whereas some bulk materials have a surface of about 1 square meter per gram, it can be as high as 600 m2/g for nanoscaled particles (7, 28). Taking this large surface into account, the chance and speed of oxidation from V2O3 to V2O5 increases dramatically and makes it, somehow, more available to cells. Thus, nanoscale vanadium oxide induces a more pronounced loss in viability, higher rate of lipid peroxidation, and an elevated oxidative stress, as indicated by HO-1 and DCF assay (see Figures 3-5). HO-1 as an indicator for oxidative stress was also used in studies on ultrafine particulate pollutants, which can induce this enzyme as well (32). Furthermore, vanadate induces ‚O2-, ‚OH, and H2O2 production in A549 cells (33). Lipid peroxidation was also investigated for vanadium bulk material, vanadyl, but not vanadate caused lipid peroxidation (34). A study from Ding and his co-workers showed that vanadium activates AP-1 via the production of the reactive oxygen species ‚O2and H2O2, but not ‚OH (35). In rats, it was shown that V2O5 induces neurotoxicity, the main areas affected were the hippocampus and the cerebellum (36). With regard to genotoxicity and DNA damage, nanoscaled materials have probably the same effects as bulk material, because nanoparticles also cause production of reactive oxygen species, which induce those effects. For vanadium oxide, either V2O3 or V2O5, teratogenic and genotoxic effects have been described (37-40). For nanoscaled vanadium oxide these mechanisms have not been investigated so far. Therefore, it is not possible to transmit these effects from data of the bulk material to nanomaterials, as nanomaterials have other properties than bulk material. As long as recognition, uptake and transport of nanoscale materials is not yet fully understood, there is much more work to be done to elucidate the specific mode of action of nano materials.

Acknowledgments This work was supported by DFG-CFN grant E1.3 and the Forschungszentrum Karlsruhe.

Supporting Information Available Western blot analysis data on HO-1 protein expression levels, comparing vanadium bulk and nano materials can be found in Figure S1. For analysis data on vanadium solubility, an ICP-OES spectra can be found in Figure S2. Solubility kinetics of different vanadium preparations from V2O3, V2O5, and V2O3 nano can be found also in Figure S3. This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review May 12, 2006. Revised manuscript received September 17, 2006. Accepted September 18, 2006. ES061140X