Toxicological Properties of Nanoparticles of Organic Compounds

Mar 6, 2009 - of Experimental Medicine, Second University of Naples,. Institute for Research on Combustion, CNR, Naples,. Department of Materials and ...
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Environ. Sci. Technol. 2009, 43, 2608–2613

Toxicological Properties of Nanoparticles of Organic Compounds (NOC) from Flames and Vehicle Exhausts L . A . S G R O , * ,† A . S I M O N E L L I , ‡ L. PASCARELLA,‡ P. MINUTOLO,§ D . G U A R N I E R I , |,⊥ N . S A N N O L O , ‡ P . N E T T I , |,⊥ A N D A . D ’ A N N A † Department of Chemical Engineering, University of Naples, “Federico II”, P. Tecchio 80, 80125 Naples, Italy, Department of Experimental Medicine, Second University of Naples, Institute for Research on Combustion, CNR, Naples, Department of Materials and Production Engineering, University of Naples, “Federico II”, and Interdisciplinary Centre of Biomedical Materials Research (CRIB)

Received December 8, 2008. Revised manuscript received February 5, 2009. Accepted February 5, 2009.

We examined the biological reactivity in vitro of nanoparticles of organic compounds (NOC) with diameters, d ) 1-3 nm, a class of combustion-generated particulate relatively unstudied compared to larger more graphitic soot particles because of their small size even though they may contribute significantly to the organic fraction of PM sampled from vehicle exhausts and urban atmospheres. We tested NOC samples collected from 2004 model vehicle emissions and laboratory flames. NOC produced a dose dependent mutagenic response in Salmonella bacteria, suggesting that NOC may add significantly to the overall mutagenicity of vehicle emissions. Incubation with peptides caused agglomeration and precipitate of the otherwise stable NOC suspension, but the chemical and/or physical nature of the NOC-peptide interactions could not be resolved. A significant cytotoxic response was measured above a critical dose of NOC in mouse embryo fibroblasts NIH3T3 cells along with possible evidence of cellular uptake by optical and confocal microscopy. The toxicological assays showed that NOC collected from flames and vehicle exhausts effectively interacted in vitro with both prokaryotic and eukaryotic cells. Differences in mutagenic potencies observed for various Salmonella strains with and without metabolic activation indicate differences in the chemical composition of NOC collected from different vehicles and flames.

Introduction Nanoparticles of organic carbon (NOC) is the name given to the smallest mode (with diameters, d ) 1-3 nm) of incipient nanoparticles formed in slightly fuel rich hydrocarbon combustion processes, which have a more organic carbon * Corresponding author phone: +39-768-2221; fax: +39-081-5963963; e-mail: [email protected]. † Department of Chemical Engineering, University of Naples. ‡ Second University of Naples. § Institute for the Study of Combustion. | Department of Materials and Production Engineering, University of Naples. ⊥ Interdisciplinary Centre of Biomedical Materials Research (CRIB). 2608

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structure compared to larger more graphitic soot particles. There is evidence that NOC comprises a significant fraction of emissions in modern combustion systems that emit low/ nondetectable amounts of soot particles (1-4). However, the atmospheric fate and toxicological properties of NOC have not yet been systematically investigated, mostly because their detection requires research level diagnostics. Atmospheric “nucleation bursts” may indicate significant concentrations of very small combustion-generated particles that are typically not detected due to their small size (d < 5 nm) and only observed following significant growth (5). The size and optical properties of organic carbon particulate matter (OC PM) measured in flames (6) are similar to those measured in the emissions of vehicles and industrial burners (1-3) and atmospheric fog (7) and rain samples (3), which implies that some portion of NOC is emitted to the atmosphere without growing in size and that human exposure to NOC is likely. Studies that compare transmission electron microscope (TEM) measurements of particulate and the detailed speciation of PAHs adhered on particulate matter formed in flames and diesel exhausts also conclude that the smallest nanoparticles emitted from diesel powered vehicles have an organic carbon structure and are combustion-generated (4). Depending on the conditions of the exhaust plume, particle coagulation may increase the size of NOC and the OC/EC ratio of emitted particles as NOC adheres to more graphitic larger particles/aggregates. Atmospheric fine particulate matter (PM) has been associated with a wide range of health problems, including cancer, allergies, asthma, inflammatory responses in the brain similar to Alzheimer’s disease, respiratory and cardiovascular disease (from chronic exposures) and death (following shortterm exposures in sensitive subjects) (8-12). Probable factors affecting PM toxicity are chemical composition, size and solubility, but precise causal relationships between measured effects and the fraction of PM responsible is still a subject of research. Several studies find that the OC fraction of atmospheric and vehicle generated PM causes a stronger toxicological response than the elemental carbon (EC, or soot) fraction of PM (13-15). It is generally well-accepted that ultrafine OC PM is mostly a result of pollution from combustion sources, but the relative role of primary OC PM (emitted from combustion sources already in the particle phase) and secondary OC PM (PM formed in atmospheric gas-to-particle reactions involving phase OC combustion products) in human and environmental health problems is not well understood. The organic fraction of PM samples is typically extracted with solvents or by oven heating so that their size and whether they were single particles or adhered to larger soot particles in the aerosol phase before the measurement is unknown. Detailed chemical analysis accounts for only about 20% of the total extracted organic material from PM samples collected from flames (16), vehicle emissions (17), and urban atmospheres (18); in flames, the remaining material, named tar, is mainly composed of combustion-generated nanoparticles of organic carbon (NOC) (16). Although the contribution of combustion generated NOC to the organic fraction of atmospheric PM is not yet well understood, the evidence that NOC is emitted from vehicles and their characteristics (small size and complex OC structure) warrants examination of their reactivity with biological systems. Here, we used a water sampling procedure (2) to procure samples of NOC isolated from soot and semivolatiles from vehicle exhausts and laboratory flames for toxicological testing. Toxicological studies examining 10.1021/es8034768 CCC: $40.75

 2009 American Chemical Society

Published on Web 03/06/2009

d < 10 nm particles have mostly examined metal oxide, graphitic carbon and nanotube particles; these studies showed that nanoparticles translocate to and affect extrarespiratory organs, including the heart, kidney, liver, and the brain (19). Only one other study in the literature examined the toxicological effects of flame generated NOC; Arenz, et al. collected NOC from laboratory propane flames and found a dose-dependent cell death on genetically engineered cells derived from human bronchus alveolar carcinoma (20). Here, we report results of in vitro toxicological screening assays that examine the mutagenicity, biological activity, and possible cellular uptake of NOC. Finally, we speculate on the chemical nature of NOC and how it may affect its interactions with other particles and biomolecules.

Materials and Methods Water samples containing concentrated levels of NOC were collected from laboratory flames and vehicle exhausts for in vitro testing, avoiding the use of potentially toxic solvents for extraction purposes. After removing larger hydrophobic soot particles that self-separate to the surface and bottom of the sample and volatile organic species by evaporation at 45 °C, the collected water samples were subjected to a series of chemical and physical measurements reported in detail in a previous work (2). The nonvolatile material remaining in the samples was found to be mostly comprised of OC PM with the same optical properties and size (1.3-2.5 nm) as flame-generated NOC (2). We collected six samples with the following NOC concentrations: two from premixed ethylene air flames (flame-1, [NOC] ) 174 µg/mL, flame-2 [NOC] ) 410 µg/mL) and four from 2004 models vehicles exhausts (diesel-1 [NOC] ) 880 µg/mL, diesel-2 [NOC] ) 2800 µg/mL, gasoline-1 [NOC] ) 550 µg/mL and gasoline-2, [NOC] ) 1450 µg/mL). In addition to NOC, significant amounts of NO3were also measured in the collected samples (2). Here, we further confirmed the removal of volatile PAHs from the NOC samples using head space/solid phase microextraction, which measured neither PAHs nor other volatile substances. Details on the sample collection, preparation and characterization methods used in this study can be found in ref 2 and are summarized in the Supporting Information (SI). In this work, we examined the toxicological properties of NOC using the Ames mutagenicity test (21), incubation with model peptides in an attempt to identify electrophilic agents in the samples (22), and observations of cellular morphology by confocal and optical microscopy and cytotoxicity following the Alamar Blue assay (23) on mouse embryo fibroblasts NIH3T3 cells. Results were quantified in dose-response relationships, and dose was calculated in terms of NOC concentrations. We varied dose by sample dilution with the appropriate medium/water mixtures for each test. Mutagenicity Tests. We tested all six samples with the Salmonella typhimurium gene mutation assay (Ames test) (21). We followed the plate-incorporation assay employing two bacterial strains: TA100 strain (hisG46, rfa, uvrB, pKM101) for base-pair substitution and the TA98 strain (hisD3052, rfa, uvrB, pKM101) for frame-shift mutations obtained from Ames, University of California. Samples were tested with and without metabolic system S9 (from lyophilized Arochlor 1254 induced male rat liver), 2 mg S9 protein/plate, to identify pro-mutagenic compounds as well as direct mutagens, respectively. Sodium azide 2 µg/plate (SIGMA) and 2-nitrofluoren 1 µg/plate (SIGMA) were used as positive controls in the bioassays without S9, for TA100 and TA98, respectively. Cyclophosphamide 5000 µg/plate (SIGMA) and 2-aminoanthracene 1 µg/plate (SIGMA) were the positive controls in the tests with S9, for TA100 and TA98, respectively. Each sample was tested in triplicate and also included one or two negative controls (treated with distilled water containing no

particles). A volume of 0.1 mL of sample was added to the test plates so that the dose, in terms of µg/plate, is 10 times less than the concentration of NOC in the test solution (in terms of µg/mL). The plates were incubated at 37 °C for 72 h in the dark. The number of His+ revertants was counted with the colony counter Cardinal (Perceptive Instruments, Suffolk, UK). The criterion for a positive test result was a clear dose-response relationship (confidence interval g 95%) and that the number of revertant colonies on the plates incubated with sample was at least twice that counted for the control plates. Following analysis procedures reported in the literature (14), we did not include nonlinear toxic responses measured at high concentrations in the leastsquares regression line fitting procedure. Peptide Tests. Given the large number of nucleophilic sites within biological macromolecules and the complexity and incomplete knowledge of the chemical structure of NOC, we used model peptides as a first step to identify adducts and electrophilic agents present in the samples that might interact with nucleophilic sites of biological macromolecules (DNA and proteins). The gasoline-1 and diesel-1 NOC samples were analyzed by liquid chromatography/ electrospray ionization/mass spectrometry (LC/ESI/MS) before and after incubation with the four different model peptides (from Bachem Biochemica, Germany). Here, we list the peptides, chosen for their reactivity with hemoglobin, their molecular weights (MW), and 3-letter amino acid sequences (underlining the most reactive nucleophilic sites): Angiotensin I (MW ) 1296.5 Da), H2NAsp-Arg-Val-Tyr-Ile-His-Pro-Phe-HisLeuCOOH, Coxsackie B3 Virus Epitope (MW)1542.7 Da) H2NGly-Pro-Val-Glu-Ile-Asp-Ala-Ile-Thr-Ala-Ala-Ile-Gly-ArgVal-Ala-CysCOOH, Alloferon I (MW)1265.3 Da) H2NHis-GlyVal-Ser-Gly-His-Gly-Val-His-GlyCOOH, Synthetic peptide free N-terminus Cysteine (MW )967.1 Da) H2NCys-Asp-ProGly-Tyr-Ile-Gly-Ser-ArgCOOH. For each test, we mixed by agitation 5 µL of an aqueous solution of peptide at 1 µg/µL with 540 µL of sample for 18 h at 37 °C, to mimic body temperature. The chromatographic profiles with and without a buffer solution (10 mM phospate) in equal volume ratios of NOC sample and buffer solution were identical. Also, we found no difference in the chromatographic spectra of NOC samples at ambient temperature and NOC samples held at 37 °C for 18 h without peptide addition. Details on the analytical procedures used for LC/ESI/MS are included in the SI. Cytotoxicity Testing and Observations of Cell Morphology. The diesel-1, gasoline-1, and flame-1 samples, initially diluted 1:20 in cell growth medium, were used to test the effects of NOC on mouse embryo fibroblasts, NIH3T3 cells. Cell viability was measured by the Alamar Blue cytotoxicity test, and cell morphology was examined with optical and confocal microscopy. Details regarding the methods of cell culture growth and treatment for observation by optical and confocal microscopy are provided in the SI. Cell viability was measured using the Alamar Blue Assay (Biosource), which incorporates an oxidation-reduction (REDOX) indicator that both fluoresces and changes color in response to a chemical reduction of growth medium resulting from cell growth (23). Cellular metabolic activity is related to the percentage of indicator reduction, which was calculated from measured absorbances at 570 and 600 nm, according to the equation provided by the supplier where 0% viability is the absorbances measured from incubated samples containing REDOX indicator diluted in cell growth medium without cells. Cell viability was correlated with the concentration of nanoparticles in the cell culture medium. After incubation with NOC, the samples were rinsed twice with PBS 1X and fixed with paraformaldeyde 4% for 20 min at room temperature. Fluorescence analyses were performed with a confocal laser scanning microscope (CLSM) Zeiss LSM 510, equipped with VOL. 43, NO. 7, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Dose-response curves for the Salmonella TA100 strain without metabolic activation, S9, for samples collected from gasoline (A) and diesel (B) powered vehicles and laboratory flames (C). The dashed horizontal lines show the response criteria for mutagenicity (double the control). Note the different x-axis scale for the NOC samples from flames (C).

FIGURE 1. Dose response curves for the Salmonella TA98 strain without metabolic activation, S9 (A, B, C) and with S9 (D). The dashed horizontal lines show the response criteria for mutagenicity (double the control). The lines connecting measurements in (D) are added only for clarity. an argon laser at a wavelength of 458 nm and objectives 40×. Images were acquired with a resolution of 512 × 512 pixel. The emitted fluorescence was detected using a high pass 475 nm filter. Since the Alamar blue reagent is fluorescent, the fluorescence microscopy experiments were carried out on different cells to avoid interference from the reagent.

Results Mutagenicity Tests. Figures 1 and 2 show the results of the Salmonella gene mutation assay, reported as the average revertant/plate counted for each concentration tested in triplicate, with (+S9) and without (-S9) metabolic activation for the TA98 (Figure 1) and TA100 (Figure 2) strains. A linear dose response was found for both strains -S9 in the higher concentration samples collected from vehicle exhausts (gasoline-2 and diesel-2 in Figures 1A-C and 2A-C). At least one tested concentration resulted mutagenic for the TA98 strain (above the dotted line, indicating twice the number of revertants counted in the controls) (Figure 1). The potency (slope of the dose-response curve) in the TA100 strain was lower, and the criteria for mutagenicity was not surpassed (with the exception of the gasoline-2 sample). A linear dose-response was also found for the flame samples, which had lower NOC concentrations than those collected from vehicle exhausts. The flame samples showed higher potencies for both strains without S9 than the samples collected from vehicle exhausts. Since only the highest tested concentration (41 µg/pl) of flame-generated NOC resulted mutagenic for 2610

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the TA98 strain (Figure 1C) and slightly below the mutagenic criteria for the TA100 strain (Figure 2C) -S9, slightly higher concentrations should be tested to better confirm mutagenicity. The samples from vehicles with lower NOC concentrations (gasoline-1 and diesel-1) resulted nonmutagenic for both strains -S9 (Figure 1A and B). Metabolic activation significantly increased the potency of the mutagenic response for the vehicle samples in tests using the TA98 strain so that the mutagenic response for the vehicle samples was observed already at the lowest concentration tested, followed by a nonlinear toxic response (Figure 1D). Different from the vehicle exhaust samples, metabolic activation reduced the potency of flame generated NOC for the TA98 strain (results not shown). Tests for all samples from flames and vehicle exhausts resulted nonmutagenic with no linear dose-response relationship for the TA100 strain +S9 (results not shown). The combined results show that NOC samples from different vehicles and flames present different mutagenic responses, which indicates that the mutagenic response may be associated with a portion of NOC with a more specific chemical structure or an interfering species present in the samples. Sulfates, which may be present in the samples from vehicles run on low-sulfur fuels, are not present in samples from sulfur-free ethylene flames. Since the mutagenic responses -S9 for NOC samples from flames had higher mutagenic potencies than those collected from vehicles, it is most likely that the measured effects are due to organic nanoparticles in the samples. Different mutagenic responses measured for samples collected from different sources likely indicate different chemical structures in the various NOC samples. Peptide Tests. LC/ESI/MS analysis of NOC samples indicates that NOC is a complex class of compounds with many unresolved peaks, in agreement with other works examining combustion-generated soot precursor material (SI Figures S1-S3) (4, 16). While the analysis did not improve our knowledge of the chemical structure of NOC, we noted

FIGURE 3. Dose-response curve of Alamar Blue assay. similar patterns in the spectra of the gasoline and diesel NOC samples, indicating similarities in their chemical nature. Example spectra by LC/ESI/MS are found in the SI. The chromatographic spectra showed numerous peaks that elute about every 5 min, with molecular weights that progressively increase with increasing retention time. Each chromatographic peak contained more than one substance, and substances present in adjacent peaks showed an increment in m/z of 44 Da. For example, the mass spectra of species eluting at 29.91 min from the gasoline NOC sample showed mainly m/z ) 283.1 and 300.1, and the adjacent peak eluting at 34.48 min showed m/z )327.1 and )344.1 (SI Figure S1). The chromatographic peaks at higher retention times showed progressively more complex mass spectra, and the pattern of several species in adjacent chromatographic peaks having an m/z spacing of 44 amu was observed in the mass range of 300-1978.7 Da (see SI Figure S2). The 44 amu spacing observed in the full scan mass spectra of NOC samples collected from vehicle exhausts may indicate functionalities containing oxygen (CO2) within the structure of NOC. The peptides effectively interacted with NOC since the peptide peaks were no longer present after incubation (SI Figure S3). The chromatographic spectra of NOC samples after incubation with model peptides were relatively similar to those of the original samples except for the partial disappearance of chromatographic peaks corresponding to NOC and a series of additional overlapping peaks with retention times longer than 60 min. The full scan mass spectra of the long retention time chromatographic peaks showed molecular weights both greater than and less than the sum of the of the peptide and NOC, which could not be attributed to a simple covalent adduct. Molecular weights less than the sum of the peptide and NOC could only be explained by an interaction of NOC particles with themselves stimulated by the presence of the peptide. Molecular weights larger than the sum of peptide and NOC could be due to a single peptide interacting with more than one NOC particle. Since adducts could not be identified, the observed increase in size of NOC observed with peptide addition may be due to chemical and/ or physical bonds. In addition to the presence of the high molecular mass structures at long retention times, we also observed a precipitate (a brownish yellow sludge at the bottom of the sample) after incubation with the peptides. Cytotoxicity Tests and Observations of Cell Morphology. Figure 3 shows that cell viability of mouse embryo fibroblast NIH3T3 cells decreased by about 20% when cells were exposed to 22 µg/mL of NOC collected from diesel exhaust and 19 µg/mL of NOC from gasoline exhaust samples. At the same NOC concentrations, cells treated with particles appeared rounded with great vacuoles inside (Figure 4B and C), while the control cells appeared well spread out on the cell medium (Figure 4A) by optical (results not shown) and confocal microscope. Control cells treated with clean NOCfree water showed no morphological changes or decrease in cell viability, suggesting that cell death does not depend on osmotic lysis.

FIGURE 4. Confocal transmission microscopy images of transmission micrographs of control cells (A) and cells treated with samples from gasoline (B) and diesel (C) powered vehicles. Confocal laser scanning fluorescence microscopy (CLS) images of control cells (D) and cells treated with samples from gasoline (E) and diesel (F) powered vehicles. To evaluate NOC uptake by cells, we analyzed our samples by fluorescence confocal microscopy since NOC particles are known to be weakly fluorescent (6). We observed weak fluorescence within the cytoplasm of untreated control cells (Figure 4D) when excited with 458 nm laser radiation. Cells treated with NOC showed more fluorescence inside the cell than control cells (Figure 4E and F), and the fluorescence was homogeneously distributed in the cytoplasm. Single particles could not be detected since the small dimensions of NOC (2 nm) are below the spatial resolution of the confocal microscopy while the size of the cells are on the order of tens of micrometers. The fluorescence was more evident for cells incubated with the diesel sample containing higher NOC concentrations than the gasoline sample in Figure 4F. The observed variation of fluorescence may be ascribed to one or both of the following phenomena: NOC internalization and activation of a cellular response to external stress (e.g., apoptosis) (24-26). Flame samples were also tested, but their concentrations were about four times lower than the vehicle samples tested. As a result, the flame samples did not show effects after 1-3 h. Tests investigating longer incubation periods showed a dose-dependent decline in cell viability after a 24 h incubation period, even at the low concentrations tested.

Discussion This work tested the biological reactivity of nanoparticles of organic carbon (NOC), a class of particles collected from flames and model 2004 vehicles that are not typically isolated for toxicological assays. Since Ames testing showed mutagenic responses of NOC on prokaryotic organisms, and a dosedependent cell viability was observed in eukaryotic (mouse fiberblast NIH3T3) cells treated with NOC, further testing on human cell lines would be worthwhile. The results presented here indicate that NOC may contribute to observed health effects related to OC PM exposure, but more work is needed to confirm this hypothesis. The mutagenic responses measured for NOC were similar in magnitude to those reported for the extracted organic fraction of PM samples collected on filters (14), suggesting that NOC may contribute to the increased mutagenicity of atmospheric particulate (27, 28). Pederson, et al. found that 20% of the total mutagenicity measured with human cell mutation assay in atmospheric OC PM samples could be related to known mutagens, most of which originated from combustion sources (28). Known mutagens include mainly nitro-PAHs, which are found in diesel exhausts and proposed as the major contributors to mutagenic responses in Ames VOL. 43, NO. 7, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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testing (29, 30) and PAHs, which are found in comparable concentrations in both gasoline and diesel exhausts (31). PAHs and their nitrated and oxygenated derivatives are reported to have a higher potency for the TA100 strain than for the TA98 strain with and without metabolic activation (32, 33). The result that NOC samples showed higher responses for the TA98 strain rather than the TA100 strain in this study may indicate that NOC and PAHs induce mutagenicity by different mechanisms. The unexplained 80% of the total mutagenicity measured in atmospheric PM samples may be due to some yet-unknown semipolar and polar mutagens or to synergistic effects between the known mutagens (28), and NOC may contribute to this yetunexplained mutagenicity. The differences observed in the mutagenic responses of flame samples compared to vehicle samples are interesting, and indicate a difference in chemical structure of NOC. The flame samples showed higher potencies for both strains without S9 than the samples collected from vehicle exhausts. Also, S9 increased the mutagenic response of vehicle generated NOC, while it decreased the potency of flame-generated NOC. Better chemical characterization of the samples is needed to correlate the observations with specific properties of the samples. Current chemical analytical methods, which usually require exorbitant sample times, are unable to fully chemically describe NOC or resolve the complex structure of OC PM samples collected from combustion exhausts or condensed from urban atmospheres. Future work should focus on quantifying the relative differences in the structure of NOC from different sources that might explain their different mutagenic potentials (like the relative amounts of aliphatic/aromatic bond structures and inclusions of oxygen/ nitrogen, and in the case of vehicle exhausts, sulfur). Methods like surface-enhanced Raman spectrometry (SERS), which recently showed the presence of oxygen in NOC samples collected from flames (36), are promising for characterizing the relative differences of NOC collected from various combustion sources. Also, tests of the ROS-forming potential of NOC samples with and without S-9 may help explain the metabolic pathway for the observed mutagenic responses. Although the covalent nature of the interaction with peptides could not be confirmed, the disappearance of the peptide signals and the appearance of unresolved peaks at high retention times and a visible precipitate in the incubated samples indicates that the NOC samples collected from vehicles interacted with all four model peptides tested and resulted in particle agglomeration. Kendall, et al. (34) also found that stable nanoparticle suspensions in laboratory saline solutions agglomerate and precipitate when suspended in lung fluid. They hypothesized that agglomeration stimulated by proteins in the lung fluid may be a mechanism for the eventual removal of very small nanoparticles from biological systems, but they do not explain how proteins aid in nanoparticle growth mechanisms. We suspect that peptides stimulate a change in the chemical structure of NOC, causing the observed agglomeration of the otherwise stable sample solution. Oxygen incorporated in the structure of graphene particles has the effect of preventing their agglomeration (35). By analogy, it is possible that interactions with peptides or proteins remove oxygen from the structure of NOC, causing the observed agglomeration. These hypotheses and observations may help direct future attempts to better characterize NOC and understand its behaviors.

Acknowledgments This work was supported by the Programma Operativo Nazionale (PON) “Ricerca Scientifica, Sviluppo Tecnologico ed Alta Formazione” 2000/2006 (Italy) and by the Health Effects Institute (HEI) contract no. 4702-RFPA03-4/03-14 (USA). The views expressed by us are our own and do not 2612

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necessarily reflect those of the supporting organizations. We thank Dr. Antonio Borghese, Istituto Motori (CNR) for the collection of samples from vehicle exhausts. We dedicate this article to the late Prof. Antonio D’Alessio. This work was the product of his vision and persistent motivation. He inspired and contributed to all stages of the work from the initial study design to the interpretations of the results, and this work would not have been possible without him.

Supporting Information Available A more detailed description of the methods used for collecting, preparing and characterizing the samples for this work that may be of interest to specialists. This material is available free of charge via the Internet at http://pubs.acs.org.

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