Raw, non-functionalized nanoparticles such as the materials used in this study form the basis for the synthesis of other functionalized particles and composite materials that are useful in both industry and medicine. As such, it is the non-functionalized forms that will be manufactured in large quantities and are, therefore, the most likely forms that will contribute to environmental contamination as well as work-place exposures. As shown in the Results these particles are very sparingly dispersible, even after sonication in a protein-rich solution such as serum. The results predict that there may be two very different types of effects of the nanoparticles on cellular response – those manifested in response to the presence of large agglomerants of particles that adhere to the surface of the epithelial cells (micro effects) and those manifested by exposure to individual particles (nanoeffects). At concentrations above 1 μg/ml (0.4 μg/cm2), both single- and multi-walled nanotubes form visible aggregates. These observations are corroborated by the dielectric spectroscopy results and AFM analysis.
In a previous study examining the effect of MWNT on human epidermal keratinocytes (Witzmann and Monteiro-Riviere 2006
), the authors observed significant alterations in protein expression, particularly in membrane-related proteins, such as those involved with organization of membrane domains and/or - membrane-cytoskeleton linkages, certain exocytotic and endocytotic transport steps, cytoskeletal integrity, and related signaling functions. A subsequent experiment in which mpkCCDcl4
cells were exposed to very high concentrations of CNPs (200 μg/cm2
) (Amos et al. 2008
) demonstrated significant declines in TEER but minor proteomic alterations. However, those proteins that were affected were related to junctional and cell adhesion functions. This leads us to hypothesize that MWNT and other related CNPs might alter barrier function in these and other epithelial cells that play such a protective role.
Studies have suggested that carbon nanoparticles may have carcinogenic properties (Murr et al. 2005
). MWNT have been specifically implicated due to their structural similarity to chrysotile asbestos that is widely accepted to cause carcinogenic responses in humans (Kane and Hurt 2008
; Poland et al. 2008
). After exposure to high concentrations of nanotubes, we observed changes in cells surrounding agglomerations of SWNT and MWNT. Cells seemed to exhibit proliferating nuclei as indicated by PCNA staining. Under control conditions, once confluence is reached, cells no longer actively divide. The results with the nanotubes indicate that SWNT and MWNT agglomerations cause cells to replicate abnormally, suggesting that hyperplasia is occurring. Despite elevated PCNA staining at agglomerant foci, PCNA expression was detected but remained unchanged at all doses analyzed using quantitative mass spectrometry.
The mpkCCDcl4 studies were conducted to show the effects of relatively short-term exposure to nanoparticles. High resistance cell lines most closely mimic the in vivo situation if potential effectors are added to the cultures after the cells have achieved a confluent monolayer, thus limiting the timing of the experiments to several days. However, the effects seen in this model could portend additional, more serious, outcomes with longer-term exposures.
In some organs, nanoparticles may remain embedded in the tissues for long periods of time. MWNT administered intratracheally persisted in the lung tissue for more than 60 days (Muller et al. 2005
). In the rodent models and in cultured lung cells, high levels of MWNT caused inflammatory and fibrotic reactions as well as evidence of mutagenesis (Muller et al. 2005
). In contrast, a single oropharyngeal aspiration of SWNT caused no inflammatory response 1 or 21 days post-exposure. However, at 21 days, the investigators did observe small interstitial fibrotic lesions in the alveolar regions, particularly near groups of macrophages containing micron-sized aggregates of the nanotubes (Mangum et al. 2006
While studies employing high concentrations of CNPs are useful for predicting effects, concentrations above 1 μg/ml are unlikely to occur in vivo except during topical exposure of keratinocytes and perhaps in lung epithelia in an industrial setting. Thus, the current studies also explored the effects of very low concentrations of nanoparticles on cellular function and protein expression in renal cells. To our knowledge these are the first studies to comprehensively examine concentrations in the low ng/ml (ng/cm2) range.
TEER is a measure of monolayer integrity and is also a very sensitive measure of cellular viability. As cellular viability decreases, TEER falls precipitously. In the experiments shown in , TEER was significantly decreased after treatment with all concentrations of MWNT from 40 μg/cm2 down to 0.4 ng/cm2. Interestingly, after incubation with fullerenes or SWNT, resistances were lowered only by exposure to concentrations below 0.4 μg/cm2.
It is important to note that none of the changes in TEER reached levels that represent a decrease in cell viability. Control monolayers had an average TEER of 1867 Ω · cm2. A decrease to values between 750 and 1000 Ω · cm2 is still considered a high resistance, intact epithelium. The changes in resistance indicate more subtle changes within the cells, and perhaps between them. Examples of cellular alterations which could be manifested in the magnitude of the changes observed would be minor modifications of the cytoskeleton which is a major component in determining the impermeability of the junctional complexes or changes in the composition of the cellular membrane which would be sufficient to alter permeability. We have not tested concentrations below those listed in so cannot yet comment on the lowest concentration that causes this change in transepithelial resistance.
To further investigate nanotube-induced changes in cellular function, the responses to external stimuli were assessed using the method of short circuit current (SCC), a measure of net ion transport. Interestingly, there were no alterations in the cellular response to ADH, a hormone known to regulate both Na+ and Cl− in the principal cells. Thus, this cellular function is maintained in the presence of nanoparticle exposure.
The proteomic analyses have highlighted several important aspects of the responses to nanoparticle exposure. The first observation is that, in general, the effects on protein expression correspond to the results of the transepithelial resistance in that they are, interestingly, inversely related to dose. Both the number of altered proteins and the fold-changes increase as dose declines. No one has previously studied carbon nanoparticle doses this low so there are no parallels in the published literature. Size measurements suggest that at high doses where there is significant agglomeration, particles act at a microscale, while at the lower doses, CNPs may act at a nanoscale level where little if any agglomeration is observed.
Second, none of the protein groups reflect a ‘traditional’ set of toxicity-related proteins. Thus, at least in the mpkCCDcl4 cells we are not observing a ‘toxic effect’ in the classic sense. This finding is in agreement with the lack of cell death in the TEER and LDH measurements and lack of substantial stress responses as measured by cytokine secretion. In fact, stress responses proteins such as those upregulated by ROS were notably unchanged by CNP exposure.
Third, there is little overlap among the three nanoparticle types in terms of the types of proteins, molecular interaction networks and pathways, suggesting the physico-chemically distinct particles act differently at the biochemical and molecular level, with respect to effects on the proteome. While the nanomaterials decreased TEER in the mpkCCDcl4 cells, the protein alterations observed do not provide evidence of mechanism in this regard. There is only one exception where SWNT at 0.04 μg/cm2 upregulated tight junction signaling proteins (). However, we have only conducted the cytoskeletal and proliferating nuclei labeling experiments at high concentrations, where the carbon forms can be located for analysis of surrounding cells, while the proteomic changes are observed predominately at the lower concentrations. Therefore additional experimentation will be necessary to fully explain the correlation, if any, between the observed changes in cellular proliferation and actin cytoskeleton structure and the proteomic changes that have been observed.
In general, the proteomic results demonstrate a complex and diverse biological response to CNP exposure, at the lowest concentrations ever studied. Furthermore, as already noted, this response is inversely related to dose, is CNP-specific, and occurs in the absence of overt irritation, inflammation, or toxicity. The inverse relationship between CNP exposure concentration and protein expression is not surprising when considered in the context of particle size. At 40 and 4 μg/cm2, significant agglomeration is evident while visible particles are absent below that level. Physico-chemical characterization evidence supports this.
When one examines mpkCCDcl4 cell proteins whose expression was altered by CNP exposure, the 40 μg/cm2 had little effect. At P < 0.01 one would expect ~ 19 proteins to be altered by chance alone, thus one can conclude this exposure was insignificant relative to protein expression, despite the decline in TEER observed in MWNT. However, at exposures several orders of magnitude lower, CNP-mediated protein expression was significantly greater and these alterations parallel significant declines in TEER. This is likely the result of CNP agglomerants (at 40 μg/cm2) exerting focal effects that may be significant in those foci, but are rendered largely undetectable when all cells on the transwell are examined. As mentioned earlier, this accounts for focal elevations of PCNA staining, but no significant change overall in PCNA as measured by label-free quantitative mass spectrometry.
As indicates, the lowest two SWNT exposures studied by proteomics resulted in a decreased expression of six ribosomal proteins. Neither C60
nor MWNT exerted such an effect. Ribosomal proteins are integral components of basic cellular machinery involved in protein synthesis and individual ribosomal proteins are known play a role in regulating cell growth, transformation and death (Warner and McIntosh 2009
). It has been suggested that both the differentiation state and the proliferative status of the cells affect the expression of ribosomal protein mRNAs (Bevort and Leffers 2000
) and both L and S-type ribosomal protein gene expression have been shown to decline in nephrotoxicity (Leussink et al. 2003
Of the proteins altered by MWNT low-dose exposure, conflicting responses were observed. The four histone family proteins were up-regulated by the 0.004 μg/cm2
exposure, while the same proteins were significantly down-regulated by the higher 0.04 μg/cm2
exposure. One of these, H2AFX, is a major component of the nucleosome core structure that comprises 10–15% of total cellular H2A family proteins in mammalian cells, is critical to genomic stability and DNA repair (Fernandez-Capetillo et al. 2004
), and play an important role in the regulation of gene expression and cellular proliferation (Svotelis et al. 2009
).The conflicting effects of MWNT on these members of the histone H2A family at these exposures are difficult to interpret but they suggest significant MWNT effects on nuclear function of these barrier epithelia.
To aid in interpretation of the complex proteomic results, we used Ingenuity Pathways Analysis. As mentioned above, protein expression was relatively unaffected by the 40 μg/cm2 exposure and no statistically significant relationships to functional networks were detected.
One of the effects observed in all three CNP exposures only at the 0.004 μg/cm2 dose was a general up-regulation of glycolysis/gluconeogenesis pathways. As indicates, expressions of proteins in this pathway were generally increased. These included enolase 2, enolase 3, and lactate dehydrogenase A (C60 exposure); aldolase A, enolase 1, glyceraldehyde-3-phosphate dehydrogenase, glucose phosphate isomerase, lactate dehydrogenase A, phosphofructokinase, phosphoglycerate kinase, phosphoglucomutase, pyruvate kinase and triosephosphate isomerase (SWNT exposure); and aldolase B, enolase 2, enolase 3, phosphoglycerate kinase, phosphoglycerate mutase, and pyruvate kinase (MWNT exposure). The only proteins associated with this pathway that were down-regulated were hexokinase 1 (C60 exposure); alcohol dehydrogenase 4 (SWNT exposure); and glyceraldehyde-3-phosphate dehydrogenase (MWNT exposure).
Gou et al. (2008) reported a dose-dependent adsorption and depletion of nutrients from RPMI cell culture medium by purified SWNTs that contained 10% Fe. HepG2 cells cultured in these depleted media showed significantly reduced viability that was restored by replenishment of folate. However, those effects occurred at doses of 10 μg/ml (corresponding to 25 μg/cm2) and above, higher than the doses used in the present study. Alternatively, one can view the increased glycolysis/gluconeogenesis pathway as a generalized response to stress. Systemically, such phenomena are usually manifested in a fight or flight response. However, the stimuli for a more subtle, analogous change at a tissue or molecular level are not well documented.
These altered components of the glycolysis/gluconeogenesis pathway are also reflected in the Functional Networks presented in . For instance, in low-dose C60 exposure, the predominant interaction network affected is a network of gene expression, cell death, and energy production. This network received a significance score of 32 (with 35 being the maximum), the highest in this exposure analysis (thus the proteins have a 1 in 1032 probability of having been assigned to this network by chance). The network contains the following differentially expressed (13 up-regulated and four down-regulated) proteins (by gene symbol): acetyl-Coenzyme A acyltransferase 2, ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1, ATP synthase, H+ transporting, mitochondrial F1 complex, beta, ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit, DEAD (Asp-Glu-Ala-Asp) box polypeptide 5, enolase 2 (gamma, neuronal), enolase 3 (beta, muscle), GTPase activating protein (SH3 domain) binding protein 2, glutathione transferase zeta 1, hexokinase 1, high-mobility group box 2, high-mobility group box 1, hydroxysteroid (17-beta) dehydrogenase 10, lactate dehydrogenase A, microtubule-associated protein, RP/EB family, member 1, phospholipase C, gamma 1, and ubiquitin-conjugating enzyme E2L 3, including members of the glycolysis/gluconeogenesis pathway. This is also observed to an even greater extent in the low-dose SWNT exposure (), where Carbohydrate Metabolism is part of the second-rated network (significance score of 20) along with Cancer and Genetic Disorder, and contains the following differentially expressed proteins: acyl-Coenzyme A binding domain containing 3, actin, alpha 1, aldolase A, fructose-bisphosphate, acidic (leucine-rich) nuclear phosphoprotein 32 family, member A, HLA-B associated transcript 1, ELAV (embryonic lethal, abnormal vision, Drosophila)-like 1 (Hu antigen R), enolase 1, (alpha), glyceraldehyde-3-phosphate dehydrogenase, glucose phosphate isomerase, heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor A), lactate dehydrogenase A, myosin, heavy chain 9, non-muscle, nucleoside diphosphate kinase, phosphoglycerate kinase 1, ribosomal protein S5, talin 1, and triosephosphate isomerase 1. Again, glycolytic/gluconeogenic proteins are interacting broadly in this functional network and are implicated in the biological effects of CNPs in this regard. As mentioned earlier, such effects are absent at exposures where agglomeration is evident. The functional networks and pathways impacted by low-dose CNP exposure are thus likely to be nanoscale effects.
Other interaction networks and pathways presented in Tables and are established in fundamentally the same manner as that described above. Only the specific protein components differ. For instance, fullerene exposure at 0.004 μg/cm2
is associated with a predominant up-regulation in the expression of proteins in cell cycle, gene expression and cell death functional networks. These, and several other functional networks, appear in bold print in to emphasize the fact that these network components are similarly affected to some degree by all three CNPs studied, at the lowest two doses. Studies of bronchoalveolar cells in mice have demonstrated increased apoptosis and GI arrest after 14 and 28 days of C60
instillation (Park et al. 2010
). That same study also showed that the expression of the tissue damagerelated MMPs, Timp and Slpi gene, and the expression of oxidative stress related SOD gene were increased until day 28 after a single instillation. Protein changes related to cell death have also been reported by SWNT exposures by Cui in HEK293 cells (Cui et al. 2005
), along with down-regulated cellular growth and proliferation. These changes were accompanied by a decrease of markers associated with G1 to S transition (e.g., cdk2, cdk4, and cyclin A, E, and D3) as well as markers associated with S, G2, and M phase and down-regulation of expression of adhesion proteins (laminin, fibronectin, cadherin FAK and collagen IV). The relevance of the changes we have observed to those alterations seen by others in various tissues will require additional studies.
Although we have no evidence of oxidative stress, as ROS-related protein alterations are otherwise absent from our data as is any other evidence of cytotoxic effect, the pathway analysis reported in suggests the SWNT-mediated up-regulation of NRF-2 mediated oxidative stress response at 0.04 μg/cm2 (in contrast to the decrease in this response at 40 μg/cm2), is consistent with an overall trend observed in our study, that the CNPs have greater biological effect at lower as opposed to higher exposure levels.
Other pathways impacted by CNP exposure at very low levels include alteration in functional networks involved in inflammatory diseases and regulation of amino acid metabolism. Changes in the expression of components of various metabolic pathways may be particle specific as well as dose- and exposure time-dependent. These aspects require further investigation.