In this inhalation study, we chose to perform our assessments using a cumulative dose - 5 mg/m3
, 5 hrs, 4 days. The dose-responses of SWCNT exposure causing pulmonary damage/toxicity in C56BL6 mice were addressed in our previous publications.6, 26
Pharyngeal aspiration exposure of mice to respirable SWCNT within the 5, 10, 20 μg SWCNT/mouse range was found to efficiently induce dose-dependent pulmonary damage, interstitial fibrosis and granulomatous lesions in mouse lungs. Even though there has been a concern that pharyngeal aspiration as a single exposure to a bolus of SWCNT may induce artificial response – it has been shown to closely mimic the inhalation exposure route and is currently accepted for assessments of health outcomes of respirable particles and infectious agents. Comparison of the pulmonary/toxicological data obtained from inhalation vs
pharyngeal aspiration studies revealed that the calculated deposited dose of SWCNT used in this inhalation study (5 mg/m3
, 5 hrs, 4 days) was 5 μg/mouse. Therefore, the exposure level in the current inhalation study was designed based on adverse outcomes found from an equivalent mass of deposited SWCNT used in pharyngeal aspiration protocols. Additionally, the rational for use of 5 mg/m3
dose for the inhalation study is based on the Permissible Exposure Level (PEL) set by OSHA for respirable synthetic graphite dust which is currently applied to SWCNT. This dose would be achieved by workers exposed for less than 2 years at the peak airborne concentrations measured in an occupational setting.34
Based on the data obtained from the inhalation study, it could be inferred that if workers were subjected to extended exposures to respirable SWCNT at current PEL for synthetic graphite, they would likely have an increased risk of pulmonary damage. The dose of SWCNT used in this inhalation study may also be compared with regulatory permissible levels established for ambient micro-particles, similar to those which the EPA established for PM2.5
(24 hrs daily) and 15 μg/m3
Some level of oral exposure and systemic distribution of SWCNT can take place during the prolonged SWCNT inhalations. In our experiments, mice were cleaned following daily inhalation exposures and then placed into new cages to minimize/avoid the potential for oral consumption of SWCNT during the night. The data regarding the oral toxicity of SWCNT in mice are scarce. Deng et al36
demonstrated that, after oral administration of multi-walled carbon nanotubes (MWCNT) (10 μg/mouse), the majority of nanoparticles remained within the stomach and small and large intestines, with no detectable transport into the blood stream. MWCNTs found in the gut remained unchanged. Similarly, no SWCNT translocation was reported by Folkmann et al
After oral administration to rats, no detectable levels of SWCNT were found in the lung and liver.
Our results demonstrate that phospholipid peroxidation in the lungs of mice exposed to SWCNT via inhalation does not exert the features typical of random free radical process but instead display high substrate and product specificity thus suggesting the involvement of enzymatic mechanisms. There are at least two important consequences from this finding: First, because non-specific radical scavenging antioxidants are not expected to be effective in suppressing enzymatically-driven lipid peroxidation, their application as protectants against SWCNT induced pulmonary damage is questionable. Second, the current observations suggest that exposure to SWCNT triggers specific pathways of cellular damage which implies that the adverse effects elicited by SWCNTs could be counteracted by targeting these pathways, likely apoptotic in nature.
Mitochondrial signaling is central to the execution of several death pathways, including apoptosis. Upon activation of mitochondria by various pro-apoptotic stimuli, cyt c, a small hemoprotein of the intermembrane space of these organelles, is released into the cytosol and acts as a co-factor for the apoptosome complex, a multi-component platform that promotes the activation of caspases, with subsequent cleavage of target proteins and dismantling of the cell.38
We have previously ascertained that cyt c can effectively catalyze peroxidation of anionic phospholipids, particularly CL, but also PS and PI.28
Moreover, this cyt c-catalyzed pathway was found to play an important role in the execution of apoptosis.6
It has been shown that CL and its oxidation products are important participants and signaling molecules in the mitochondria-dependent apoptotic cell death program.39
Early in apoptosis, massive membrane translocations of CL result in the appearance of CL in the outer mitochondrial membrane.39
Consequently, significant amounts of CL become available for the interactions with cyt c, one of the intermembrane space proteins. Binding of CL with cyt c yields a complex that acts as a potent CL-specific peroxidase40
and generates CL peroxidation products that are important for the mitochondria permeabilization and the release of cyt c and other pro-apoptotic factors form mitochondria into the cytosol.39
Given that the exposure to SWCNT triggers the peroxidation of CL in the lungs of exposed animals, one may surmise that mitochondrial apoptotic signaling is engaged in pulmonary response to SWCNT exposure. In support of this contention, the numbers of apoptotic cells in the lungs of exposed animals paralleled the selective peroxidation of lipids. Indeed, significant accumulation of apoptotic cells was observed on days 1 and 7 after SWCNT exposure, as determined by TUNEL staining.
The detection of increased numbers of apoptotic cells in vivo
could in principle be explained by an excessive rate of apoptosis and/or a decreased capacity for macrophage clearance of apoptotic cell corpses. These observations are in line with the outcome of our oxidative lipidomics assessment that showed the selective peroxidation of PS and CL (and PI), while the more abundant phospholipids PC and PE were not affected upon pulmonary exposure to SWCNT. Moreover, this interpretation is supported by our model experiments with the mouse lung phospholipids incubated with cyt c and H2
. These experiments demonstrate a very similar pattern of selectivity with predominant peroxidation of the same molecular species of CL, PS and PI as in mice exposed to SWCNT. In other words, it is possible that mitochondrial apoptosis with cyt c-driven peroxidation of CL and PS could explain the observed pattern of lipid peroxidation in the lungs of mice exposed to SWCNT. However, we cannot exclude the involvement of NADPH oxidase-derived ROS, particularly at later stages of the inflammatory response. In a previous study, we showed that NADPH oxidase-deficient mice displayed increased and persistent accumulation of neutrophils and elevated levels of apoptotic cells in the lungs, and significantly decreased fibrotic responses to SWCNT when compared to wild-type mice.7
This suggests a role for NADPH oxidase-derived ROS in determining the course of pulmonary response to SWCNT.
Our previous work has identified the enzymatic process of CL peroxidation as a potentially important target for the discovery of mitochondria-targeted anti-apoptotic small molecule protectors.41, 42
Two different types of these molecules have been designed: GS-nitroxides - conjugates of stable nitroxide radicals with mitochondria-targeting hemigramicidin S – and TPP-IFFAs – conjugates of imidazole-substituted fatty acids (oleic and stearic) with mitochondria-targeting triphenyl-phosphonium. GS-nitroxides acts as electron-scavengers effective in preventing the production of superoxide radicals and its dismutation product, H2
, in mitochondria. Because H2
feeds the peroxidase cycle involved in the catalysis of CL peroxidation, GS-nitroxides turned to be effective in inhibiting CL peroxidation.41,43
TPP-IFFAs act through a different mechanism – they are strong ligands of heme-iron in cyt c/CL complexes that block their peroxidase activity towards peroxidation of CL. Both of these newly designed small molecules regulators displayed pronounced anti-apoptotic properties in vitro
and in vivo
, for example as protectors/mitigators against apoptotic damage44
and acute radiation syndrome induced by total body irradiation.45
Assuming that apoptosis is an essential contributor to the SWCNT induced lung injury we suggest that our discovered specificity of CL peroxidation indicates a possibility of using the mitochondria-targeted GS-nitroxides and TPP-IFFAs as potentially useful regulators of CL peroxidation and anti-apoptotic protectors in the lung.
Following the induction of cell death by apoptosis, cell corpses are removed by professional phagocytes or neighboring cells in order to avoid tissue damage which would otherwise occur due to the persistent exposure to cell debris and the ensuing leakage of noxious intracellular constituents. In particular, macrophage clearance of apoptotic neutrophils at sites of inflammation is believed to be required for the effective resolution of the inflammatory process46
. Released cyt c forms cyt c/PS complexes that act as major catalysts of PS oxidation and subsequent PS externalization during late stages of apoptosis.47, 48
Externalized PS and its peroxidation products are essential “eat-me” signals for macrophage engulfment and clearance of apoptotic cells (reviewed in49
). We and others have shown that the externalization of oxidized PS on the surface of apoptotic cells is required for their effective clearance by macrophages.50-52
Moreover, the absence of PS oxidation and exposure in neutrophils derived from patients with chronic granulomatous disease (CGD) leads to the ineffective clearance by macrophages, suggesting a critical role for PS exposure and oxidation in the prevention of chronic inflammation in humans.53
The current observation of specific peroxidation of PS in the lungs of exposed mice suggests that the pathways of PS-dependent clearance of apoptotic cells may be important in tissue responses to SWCNT. It is also tempting to speculate that oxygenated species of CL, PS and PI are essential precursors of inflammatory regulators such as resolvins.15, 16
Indeed, small amounts of oxygenated docosahexaenoic acid-containing species of PS – potential substrates of PLA2
have been detected after SWCNT exposure.
Using similar oxidative lipidomics protocols, we have previously characterized the pattern of phospholipid peroxidation following several other insults, including hyperoxic acute lung injury, γ-radiation-induced lung injury, and traumatic brain injury.21, 24, 29
Strikingly, in all cases, a selective pattern of phospholipid peroxidation involving primarily CL and/or PS was observed. The fact that inhalation exposure to SWCNT with high content of transition metals, particularly iron, known to effectively catalyze non-specific peroxidation of polyunsaturated phospholipids, also induces a similar, selective pattern of peroxidation of phospholipids suggests that common pathways of cellular and tissue damage are engaged, thus suggesting that SWCNT do not elicit “nano-specific” effects. This is encouraging because it implies that common “anti-apoptotic” counter-measures against a diverse range of insults, including nanomaterials, may be envisioned. The manufacturing of nanotube material relies on the use of transition metal catalysts. Most of the iron is present in elemental form, but its redox active fraction in ionic form within carbonaceous particles may act as a catalyst of oxidative stress in biological settings. The major toxicity mechanisms induced by SWCNT include inflammatory response and oxidative stress. Because inflammatory cells generate superoxide radicals and their dismutation product, H2
– this provides a redox environment in which transition metals can fully realize their pro-oxidant potential, thus synergistically enhancing damage of iron-containing SWCNT to cells and trigger cell death programs. Our previous work has characterized apoptosis-associated phopholipid peroxidation and revealed its selectivity in predominant peroxidation of two anionic phospholipids, CL and PS.39, 47
This selectivity was due to the central catalytic role of mitochondrial cyt c capable of forming peroxidase complexes with CL in mitochondria and with PS in extramitochondrial compartments of cells. SWCNT-associated iron may also directly
induce lipid peroxidation reactions in cells and biofluids. As a random catalytic process, this reaction prefers the most abundant phospholipids with polyunsaturated fatty acid residues independently of their polar heads such as PC and PE. Notably, we found accumulation of peroxidized molecular species mostly in anionic phospholipids, CL and PS, while PC and PE remained essentially non-oxidized. This suggests that apoptosis-associated phospholipid peroxidation process was dominant while random catalytic reactions were a relatively minor contributor to the overall peroxidation. Interestingly, similar relationships were established in another type of pro-oxidant environment – total body irradiation, a well known inducer of free radicals, - free radicals, - whereby selective accumulation of peroxidation products in the lung was also detected only in CL and PS (but not in PC and PE).24
In conclusion, using global oxidative lipidomics, we characterized, for the first time, the specific peroxidation profiles of cellular phospholipids in the lungs of mice exposed to non-purified (iron-containing) SWCNT. This study thus indicates that the exposure to SWCNT results in a selective lipid peroxidation rather than in non-specific free radical oxidation.55, 56
Furthermore, the fact that we could detect specific peroxidation of CL and PS, and a concurrent elevation in the number of apoptotic cells, suggests the involvement of mitochondria-dependent apoptosis as well as macrophage disposal of apoptotic cells in the regulation of the inflammatory response to SWCNT. Further studies are warranted to uncover the source(s) of selective, likely enzymatic, lipid peroxidation triggered by exposure to SWCNT. In addition, oxidative lipidomics protocols may be applied to the study of other engineered nanomaterials, in the lung or in other tissues.