Formation of protein adducts with Hydroperoxy-PE electrophilic cleavage products during ferroptosis.

Ferroptosis is an iron dependent form of cell death, that is triggered by the discoordination of iron, lipids, and thiols. Its unique signature that distinguishes it from other forms of cell death is the formation and accumulation of lipid hydroperoxides, particularly oxidized forms of polyunsaturated phosphatidylethanolamines (PEs), which drives cell death. These readily undergo iron-catalyzed secondary free radical reactions leading to truncated products which retain the signature PE headgroup and which can readily react with nucleophilic moieties in proteins via their truncated electrophilic acyl chains. Using a redox lipidomics approach, we have identified oxidatively-truncated PE species (trPEox) in enzymatic and non-enzymatic model systems. Further, using a model peptide we demonstrate adduct formation with Cys as the preferred nucleophilic residue and PE(26:2) +2 oxygens, as one of the most reactive truncated PE-electrophiles produced. In cells stimulated to undergo ferroptosis we identified PE-truncated species with sn-2 truncations ranging from 5 to 9 carbons. Taking advantage of the free PE headgroup, we have developed a new technology using the lantibiotic duramycin, to enrich and identify the PE-lipoxidated proteins. Our results indicate that several dozens of proteins for each cell type, are PE-lipoxidated in HT-22, MLE, and H9c2 cells and M2 macrophages after they were induced to undergo ferroptosis. Pretreatment of cells with the strong nucleophile, 2-mercaptoethanol, prevented the formation of PE-lipoxidated proteins and blocked ferroptotic death. Finally, our docking simulations showed that the truncated PE species bound at least as good to several of the lantibiotic-identified proteins, as compared to the non-truncated parent molecule, stearoyl-arachidonoyl PE (SAPE), indicating that these oxidatively-truncated species favor/promote the formation of PEox-protein adducts. The identification of PEox-protein adducts during ferroptosis suggests that they are participants in the ferroptotic process preventable by 2-mercaptoethanol and may contribute to a point of no return in the ferroptotic death process.

Differential responses of murine alveolar macrophages to elongate mineral particles of asbestiform and non-asbestiform varieties: Cytotoxicity, cytokine secretion and transcriptional changes.

Human exposures to asbestiform elongate mineral particles (EMP) may lead to diffuse fibrosis, lung cancer, malignant mesothelioma and autoimmune diseases. Cleavage fragments (CF) are chemically identical to asbestiform varieties (or habits) of the parent mineral, but no consensus exists on whether to treat them as asbestos from toxicological and regulatory standpoints. Alveolar macrophages (AM) are the first responders to inhaled particulates, participating in clearance and activating other resident and recruited immunocompetent cells, impacting the long-term outcomes. In this study we address how EMP of asbestiform versus non-asbestiform habit affect AM responses. Max Planck Institute (MPI) cells, a non-transformed mouse line that has an AM phenotype and genotype, were treated with mass-, surface area- (s.a.), and particle number- (p.n.) equivalent concentrations of respirable asbestiform and non-asbestiform riebeckite/tremolite EMP for 24 h. Cytotoxicity, cytokines secretion and transcriptional changes were evaluated. At the equal mass, asbestiform EMP were more cytotoxic, however EMP of both habits induced similar LDH leakage and decrease in viability at s.a. and p.n. equivalent doses. DNA damage assessment and cell cycle analysis revealed differences in the modes of cell death between asbestos and respective CF. There was an increase in chemokines, but not pro-inflammatory cytokines after all EMP treatments. Principal component analysis of the cytokine secretion showed close clustering for the s.a. and p.n. equivalent treatments. There were mineral- and habit-specific patterns of gene expression dysregulation at s.a. equivalent doses. Our study reveals the critical nature of EMP morphometric parameters for exposure assessment and dosing approaches used in toxicity studies.

Enhanced morphological transformation of human lung epithelial cells by continuous exposure to cellulose nanocrystals.

Cellulose nanocrystals (CNC), also known as nanowhiskers, have recently gained much attention due to their biodegradable nature, advantageous chemical and mechanical properties, economic value and renewability thus making them attractive for a wide range of applications. However, before these materials can be considered for potential uses, investigation of their toxicity is prudent. Although CNC exposures are associated with pulmonary inflammation and damage as well as oxidative stress responses and genotoxicity in vivo, studies evaluating cell transformation or tumorigenic potential of CNC's were not previously conducted. In this study, we aimed to assess the neoplastic-like transformation potential of two forms of CNC derived from wood (powder and gel) in human pulmonary epithelial cells (BEAS-2B) in comparison to fibrous tremolite (TF), known to induce lung cancer. Short-term exposure to CNC or TF induced intracellular ROS increase and DNA damage while long-term exposure resulted in neoplastic-like transformation demonstrated by increased cell proliferation, anchorage-independent growth, migration and invasion. The increased proliferative responses were also in-agreement with observed levels of pro-inflammatory cytokines. Based on the hierarchical clustering analysis (HCA) of the inflammatory cytokine responses, CNC powder was segregated from the control and CNC-gel samples. This suggests that CNC may have the ability to influence neoplastic-like transformation events in pulmonary epithelial cells and that such effects are dependent on the type/form of CNC. Further studies focusing on determining and understanding molecular mechanisms underlying potential CNC cell transformation events and their likelihood to induce tumorigenic effects in vivo are highly warranted.

Redox phospholipidomics of enzymatically generated oxygenated phospholipids as specific signals of programmed cell death.

High fidelity and effective adaptive changes of the cell and tissue metabolism to changing environments require strict coordination of numerous biological processes. Multicellular organisms developed sophisticated signaling systems of monitoring and responding to these different contexts. Among these systems, oxygenated lipids play a significant role realized via a variety of re-programming mechanisms. Some of them are enacted as a part of pro-survival pathways that eliminate harmful or unnecessary molecules or organelles by a variety of degradation/hydrolytic reactions or specialized autophageal processes. When these "partial" intracellular measures are insufficient, the programs of cells death are triggered with the aim to remove irreparably damaged members of the multicellular community. These regulated cell death mechanisms are believed to heavily rely on signaling by a highly diversified group of molecules, oxygenated phospholipids (PLox). Out of thousands of detectable individual PLox species, redox phospholipidomics deciphered several specific molecules that seem to be diagnostic of specialized death programs. Oxygenated cardiolipins (CLs) and phosphatidylethanolamines (PEs) have been identified as predictive biomarkers of apoptosis and ferroptosis, respectively. This has led to decoding of the enzymatic mechanisms of their formation involving mitochondrial oxidation of CLs by cytochrome c and endoplasmic reticulum-associated oxidation of PE by lipoxygenases. Understanding of the specific biochemical radical-mediated mechanisms of these oxidative reactions opens new avenues for the design and search of highly specific regulators of cell death programs. This review emphasizes the usefulness of such selective lipid peroxidation mechanisms in contrast to the concept of random poorly controlled free radical reactions as instruments of non-specific damage of cells and their membranes. Detailed analysis of two specific examples of phospholipid oxidative signaling in apoptosis and ferroptosis along with their molecular mechanisms and roles in reprogramming has been presented.

Iron catalysis of lipid peroxidation in ferroptosis: Regulated enzymatic or random free radical reaction?

Duality of iron as an essential cofactor of many enzymatic metabolic processes and as a catalyst of poorly controlled redox-cycling reactions defines its possible biological beneficial and hazardous role in the body. In this review, we discuss these two "faces" of iron in a newly conceptualized program of regulated cell death, ferroptosis. Ferroptosis is a genetically programmed iron-dependent form of regulated cell death driven by enhanced lipid peroxidation and insufficient capacity of thiol-dependent mechanisms (glutathione peroxidase 4, GPX4) to eliminate hydroperoxy-lipids. We present arguments favoring the enzymatic mechanisms of ferroptotically engaged non-heme iron of 15-lipoxygenases (15-LOX) in complexes with phosphatidylethanolamine binding protein 1 (PEBP1) as a catalyst of highly selective and specific oxidation reactions of arachidonoyl- (AA) and adrenoyl-phosphatidylethanolamines (PE). We discuss possible role of iron chaperons as control mechanisms for guided iron delivery directly to their "protein clients" thus limiting non-enzymatic redox-cycling reactions. We also consider opportunities of loosely-bound iron to contribute to the production of pro-ferroptotic lipid oxidation products. Finally, we propose a two-stage iron-dependent mechanism for iron in ferroptosis by combining its catalytic role in the 15-LOX-driven production of 15-hydroperoxy-AA-PE (HOO-AA-PE) as well as possible involvement of loosely-bound iron in oxidative cleavage of HOO-AA-PE to oxidatively truncated electrophiles capable of attacking nucleophilic targets in yet to be identified proteins leading to cell demise.

Assessment of Airborn Multiwalled Carbon Nanotubes in a Manufactoring Environment.

This study was carried out in a factory producing multiwalled carbon nanotubes (MWCNTs) by the catalytic chemical vapor deposition method in a pyrolysis reactor. Air samples of the personal breathing areas were collected simultaneously on mixed cellulose ester filters, for analysis by transmission electron microscopy (TEM), and on high-purity quartz filters for thermal-optical analysis of elemental carbon (EC). It is found that the production of MWCNTs is accompanied by the release of the MWCNT structures in the air of different working zones. The concentration of respirable aerosol in the personal breathing areas, averaged over an 8-hour period, ranges from 0.54 to 6.11 µg/m3 based on EC. Airborne MWCNTs were found in the form of agglomerates that range in size from about 1 to 10 µm. These data are consistent with measurements in different plants by two other international groups (from the United States and Sweden) using similar methodology (TEM in combination with EC analysis). In the absence of convincing data on the potential health risks of MWCNTs, and following the principle of reasonable precautions, preventive measures should be taken to minimize exposure to these materials.

Carbon Nanotubes Exposure Risk Assessment: From Toxicology to Epidemiologic Studies (Overview of the Current Problem).

Nanoscale size and fiber like structure of carbon nanotubes (CNTs) may determine high reactivity and penetration, as well as the pathogenicity of asbestos and other mineral fibers. Despite many in vitro and in vivo studies, the absence of full-scale data on CNT effects on human health clearly point out the necessity for epidemiological studies. Currently, several projects are initiated worldwide on studying health risks associated with the inhalation of industrial CNTs, including NIOSH-promoted research (United States), the European CANTES study, and the Russian CNT-ERA project. Studies comprising several successive steps, such as CNT exposure assessment in occupational settings, toxicological evaluation, and epidemiological observations, are critical for determining material safety and use criteria.

[Hygienic evaluation of multilayer carbon nanotubes].

The authors demonstrate that traditional methods evaluating work conditions on contemporary innovative enterprises producing nanomaterials assess these conditions as harmless and safe. At the same time, special investigation methods enable to reveal new hazards for workers' health: the study results prove that workers engaged into multilayer carbon nanotubes production are exposed to multilayer carbon nanotubes aerosols in concentrations exceeding internationally acceptable levels of 1 µg/ml (NIOSH)--that can harm the workers' health.

Evaluation of fibrogenic potential of industrial multi-walled carbon nanotubes in acute aspiration experiment.

Local inflammatory response in the lungs and fi brogenic potential of multi-walled carbon nanotubes were studied in an acute aspiration experiment in mice. The doses were chosen based on the concentration of nanotubes in the air at a workplace of the company-producer. ELISA, fl ow cytometry, enhanced darkfield microscopy, and histological examination showed that multi-walled carbon nanotubes induced local inflammation, oxidative stress, and connective tissue growth (fibrosis). Serum levels of TGF-ß1 and osteopontin proteins can serve as potential exposure biomarkers.

ESR evidence for in vivo formation of free radicals in tissue of mice exposed to single-walled carbon nanotubes.

Nanomaterials are being utilized in an increasing variety of manufactured goods. Because of their unique physicochemical, electrical, mechanical, and thermal properties, single-walled carbon nanotubes (SWCNTs) have found numerous applications in the electronics, aerospace, chemical, polymer, and pharmaceutical industries. Previously, we have reported that pharyngeal exposure of C57BL/6 mice to SWCNTs caused dose-dependent formation of granulomatous bronchial interstitial pneumonia, fibrosis, oxidative stress, acute inflammatory/cytokine responses, and a decrease in pulmonary function. In the current study, we used electron spin resonance (ESR) to directly assess whether exposure to respirable SWCNTs caused formation of free radicals in the lungs and in two distant organs, the heart and liver. Here we report that exposure to partially purified SWCNTs (HiPco technique, Carbon Nanotechnologies, Inc., Houston, TX, USA) resulted in the augmentation of oxidative stress as evidenced by ESR detection of α-(4-pyridyl-1-oxide)-N-tert-butylnitrone spin-trapped carbon-centered lipid-derived radicals recorded shortly after the treatment. This was accompanied by a significant depletion of antioxidants and elevated biomarkers of inflammation presented by recruitment of inflammatory cells and an increase in proinflammatory cytokines in the lungs, as well as development of multifocal granulomatous pneumonia, interstitial fibrosis, and suppressed pulmonary function. Moreover, pulmonary exposure to SWCNTs also caused the formation of carbon-centered lipid-derived radicals in the heart and liver at later time points (day 7 postexposure). Additionally, SWCNTs induced a significant accumulation of oxidatively modified proteins, increase in lipid peroxidation products, depletion of antioxidants, and inflammatory response in both the heart and the liver. Furthermore, the iron chelator deferoxamine noticeably reduced lung inflammation and oxidative stress, indicating an important role for metal-catalyzed species in lung injury caused by SWCNTs. Overall, we provide direct evidence that lipid-derived free radicals are a critical contributor to tissue damage induced by SWCNTs not only in the lungs, but also in distant organs.

Single-walled carbon nanotube-induced mitotic disruption.

Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96 µg/cm(2) single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24-72 h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24 µg/cm(2) SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is an early event in the progression of many cancers, suggesting that it may play a role in both tumorigenesis and tumor progression. These results suggest caution should be used in the handling and processing of carbon nanotubes.

Genotoxicity of carbon nanofibers: are they potentially more or less dangerous than carbon nanotubes or asbestos?

The production of carbon nanofibers and nanotubes (CNF/CNT) and their composite products is increasing globally. CNF are generating great interest in industrial sectors such as energy production and electronics, where alternative materials may have limited performance or are produced at a much higher cost. However, despite the increasing industrial use of carbon nanofibers, information on their potential adverse health effects is limited. In the current study, we examine the cytotoxic and genotoxic potential of carbon-based nanofibers (Pyrograf®-III) and compare this material with the effects of asbestos fibers (crocidolite) or single-walled carbon nanotubes (SWCNT). The genotoxic effects in the lung fibroblast (V79) cell line were examined using two complementary assays: the comet assay and micronucleus (MN) test. In addition, we utilized fluorescence in situ hybridization to detect the chromatin pan-centromeric signals within the MN indicating their origin by aneugenic (chromosomal malsegregation) or clastogenic (chromosome breakage) mechanisms. Cytotoxicity tests revealed a concentration- and time-dependent loss of V79 cell viability after exposure to all tested materials in the following sequence: asbestos>CNF>SWCNT. Additionally, cellular uptake and generation of oxygen radicals was seen in the murine RAW264.7 macrophages following exposure to CNF or asbestos but not after administration of SWCNT. DNA damage and MN induction were found after exposure to all tested materials with the strongest effect seen for CNF. Finally, we demonstrated that CNF induced predominantly centromere-positive MN in primary human small airway epithelial cells (SAEC) indicating aneugenic events. Further investigations are warranted to elucidate the possible mechanisms involved in CNF-induced genotoxicity.

The role of nanotoxicology in realizing the 'helping without harm' paradigm of nanomedicine: lessons from studies of pulmonary effects of single-walled carbon nanotubes.

Nano-sized materials and nano-scaled processes are widely used in many industries. They are being actively introduced as diagnostic and therapeutic in biomedicine and they are found in numerous consumer products. The small size of nanoparticles, comparable with molecular machinery of cells, may affect normal physiological functions of cells and cause cytotoxicity. Their toxic potential cannot be extrapolated from studies of larger particles due to unique physicochemical properties of nanomaterials. Therefore, the use of nanomaterials may pose unknown risks to human health and the environment. This review discusses several important issues relevant to pulmonary toxicity of nanoparticles, especially single-walled carbon nanotubes (SWCNT), their direct cytotoxic effects, their ability to cause an inflammatory response, and induce oxidative stress upon pharyngeal aspiration or inhalation. Further, recognition and engulfment of nanotubes by macrophages as they relate to phagocytosis and bio-distribution of nanotubes in tissues and circulation are discussed. The immunosuppressive effects of CNT and their significance in increased sensitivity of exposed individuals to microbial infections are summarized. Finally, data on biodegradation of SWCNT by oxidative enzymes of inflammatory cells are presented in lieu of their persistence and distribution in the body.

Size-dependent effects of tungsten carbide-cobalt particles on oxygen radical production and activation of cell signaling pathways in murine epidermal cells.

Hard metal or cemented carbide consists of a mixture of tungsten carbide (WC) (85%) and metallic cobalt (Co) (5-15%). WC-Co is considered to be potentially carcinogenic to humans. However, no comparison of the adverse effects of nano-sized WC-Co particles is available to date. In the present study, we compared the ability of nano- and fine-sized WC-Co particles to form free radicals and propensity to activate the transcription factors, AP-1 and NF-kappaB, along with stimulation of mitogen-activated protein kinase (MAPK) signaling pathways in a mouse epidermal cell line (JB6 P(+)). Our results demonstrated that nano-WC-Co generated a higher level of hydroxyl radicals, induced greater oxidative stress, as evidenced by a decrease of GSH levels, and caused faster JB6 P(+) cell growth/proliferation than observed after exposure of cells to fine WC-Co. In addition, nano-WC-Co activated AP-1 and NF-kappaB more efficiently in JB6(+/+) cells as compared to fine WC-Co. Experiments using AP-1-luciferase reporter transgenic mice confirmed the activation of AP-1 by nano-WC-Co. Nano- and fine-sized WC-Co particles also stimulated MAPKs, including ERKs, p38, and JNKs with significantly higher potency of nano-WC-Co. Finally, co-incubation of the JB6(+/+) cells with N-acetyl-cysteine decreased AP-1 activation and phosphorylation of ERKs, p38 kinase, and JNKs, thus suggesting that oxidative stress is involved in WC-Co-induced toxicity and AP-1 activation.

Induction of aneuploidy by single-walled carbon nanotubes.

Engineered carbon nanotubes are newly emerging manufactured particles with potential applications in electronics, computers, aerospace, and medicine. The low density and small size of these biologically persistent particles makes respiratory exposures to workers likely during the production or use of commercial products. The narrow diameter and great length of single-walled carbon nanotubes (SWCNT) suggest the potential to interact with critical biological structures. To examine the potential of nanotubes to induce genetic damage in normal lung cells, cultured primary and immortalized human airway epithelial cells were exposed to SWCNT or a positive control, vanadium pentoxide. After 24 hr of exposure to either SWCNT or vanadium pentoxide, fragmented centrosomes, multiple mitotic spindle poles, anaphase bridges, and aneuploid chromosome number were observed. Confocal microscopy demonstrated nanotubes within the nucleus that were in association with cellular and mitotic tubulin as well as the chromatin. Our results are the first to report disruption of the mitotic spindle by SWCNT. The nanotube bundles are similar to the size of microtubules that form the mitotic spindle and may be incorporated into the mitotic spindle apparatus.

Oxidative stress and inflammatory response in dermal toxicity of single-walled carbon nanotubes.

Single-walled carbon nanotubes (SWCNT) represent a novel material with unique electronic and mechanical properties. The extremely small size ( approximately 1 nm diameter) renders their chemical and physical properties unique. A variety of different techniques are available for the production of SWCNT; however, the most common is via the disproportionation of gaseous carbon molecules supported on catalytic iron particles (high-pressure CO conversion, HiPCO). The physical nature of SWCNT may lead to dermal penetration following deposition on exposed skin. This dermal deposition provides a route of exposure which is important to consider when evaluating SWCNT toxicity. The dermal effects of SWCNT are largely unknown. We hypothesize that SWCNT may be toxic to the skin. We further hypothesize that SWCNT toxicity may be dependent upon the metal (particularly iron) content of SWCNT via the metal's ability to interact with the skin, initiate oxidative stress, and induce redox-sensitive transcription factors thereby affecting/leading to inflammation. To test this hypothesis, the effects of SWCNT were assessed both in vitro and in vivo using EpiDerm FT engineered skin, murine epidermal cells (JB6 P+), and immune-competent hairless SKH-1 mice. Engineered skin exposed to SWCNT showed increased epidermal thickness and accumulation and activation of dermal fibroblasts which resulted in increased collagen as well as release of pro-inflammatory cytokines. Exposure of JB6 P+ cells to unpurified SWCNT (30% iron) resulted in the production of ESR detectable hydroxyl radicals and caused a significant dose-dependent activation of AP-1. No significant changes in AP-1 activation were detected when partially purified SWCNT (0.23% iron) were introduced to the cells. However, NFkappaB was activated in a dose-dependent fashion by exposure to both unpurified and partially purified SWCNT. Topical exposure of SKH-1 mice (5 days, with daily doses of 40 microg/mouse, 80 microg/mouse, or 160 microug/mouse) to unpurified SWCNT caused oxidative stress, depletion of glutathione, oxidation of protein thiols and carbonyls, elevated myeloperoxidase activity, an increase of dermal cell numbers, and skin thickening resulting from the accumulation of polymorphonuclear leukocytes (PMNs) and mast cells. Altogether, these data indicated that topical exposure to unpurified SWCNT, induced free radical generation, oxidative stress, and inflammation, thus causing dermal toxicity.

Mechanisms of pulmonary toxicity and medical applications of carbon nanotubes: Two faces of Janus?

Nanotechnology is an emerging science involving manipulation of materials at the nanometer scale. There are several exciting prospects for the application of engineered nanomaterials in medicine. However, concerns over adverse and unanticipated effects on human health have also been raised. In fact, the same properties that make engineered nanomaterials attractive from a technological and biomedical perspective could also make these novel materials harmful to human health and the environment. Carbon nanotubes are cylinders of one or several coaxial graphite layer(s) with a diameter in the order of nanometers, and serve as an instructive example of the Janus-like properties of nanomaterials. Numerous in vitro and in vivo studies have shown that carbon nanotubes and/or associated contaminants or catalytic materials that arise during the production process may induce oxidative stress and prominent pulmonary inflammation. Recent studies also suggest some similarities between the pathogenic properties of multi-walled carbon nanotubes and those of asbestos fibers. On the other hand, carbon nanotubes can be readily functionalized and several studies on the use of carbon nanotubes as versatile excipients for drug delivery and imaging of disease processes have been reported, suggesting that carbon nanotubes may have a place in the armamentarium for treatment and monitoring of cancer, infection, and other disease conditions. Nanomedicine is an emerging field that holds great promise; however, close attention to safety issues is required to ensure that the opportunities that carbon nanotubes and other engineered nanoparticles offer can be translated into feasible and safe constructs for the treatment of human disease.

Inhalation vs. aspiration of single-walled carbon nanotubes in C57BL/6 mice: inflammation, fibrosis, oxidative stress, and mutagenesis.

Nanomaterials are frontier technological products used in different manufactured goods. Because of their unique physicochemical, electrical, mechanical, and thermal properties, single-walled carbon nanotubes (SWCNT) are finding numerous applications in electronics, aerospace devices, computers, and chemical, polymer, and pharmaceutical industries. SWCNT are relatively recently discovered members of the carbon allotropes that are similar in structure to fullerenes and graphite. Previously, we (47) have reported that pharyngeal aspiration of purified SWCNT by C57BL/6 mice caused dose-dependent granulomatous pneumonia, oxidative stress, acute inflammatory/cytokine responses, fibrosis, and decrease in pulmonary function. To avoid potential artifactual effects due to instillation/agglomeration associated with SWCNT, we conducted inhalation exposures using stable and uniform SWCNT dispersions obtained by a newly developed aerosolization technique (2). The inhalation of nonpurified SWCNT (iron content of 17.7% by weight) at 5 mg/m(3), 5 h/day for 4 days was compared with pharyngeal aspiration of varying doses (5-20 microg per mouse) of the same SWCNT. The chain of pathological events in both exposure routes was realized through synergized interactions of early inflammatory response and oxidative stress culminating in the development of multifocal granulomatous pneumonia and interstitial fibrosis. SWCNT inhalation was more effective than aspiration in causing inflammatory response, oxidative stress, collagen deposition, and fibrosis as well as mutations of K-ras gene locus in the lung of C57BL/6 mice.

Increased accumulation of neutrophils and decreased fibrosis in the lung of NADPH oxidase-deficient C57BL/6 mice exposed to carbon nanotubes.

Single-walled carbon nanotubes (SWCNT) have been introduced into a large number of new technologies and consumer products. The combination of their exceptional features with very broad applications raised concerns regarding their potential health effects. The prime target for SWCNT toxicity is believed to be the lung where exposure may occur through inhalation, particularly in occupational settings. Our previous work has demonstrated that SWCNT cause robust inflammatory responses in rodents with very early termination of the acute phase and rapid onset of chronic fibrosis. Timely elimination of polymorphonuclear neutrophils (PMNs) through apoptosis and their subsequent clearance by macrophages is a necessary stage in the resolution of pulmonary inflammation whereby NADPH oxidase contributes to control of apoptotic cell death and clearance of PMNs. Thus, we hypothesized that NADPH oxidase may be an important regulator of the transition from the acute inflammation to the chronic fibrotic stage in response to SWCNT. To experimentally address the hypothesis, we employed NADPH oxidase-deficient mice which lack the gp91(phox) subunit of the enzymatic complex. We found that NADPH oxidase null mice responded to SWCNT exposure with a marked accumulation of PMNs and elevated levels of apoptotic cells in the lungs, production of pro-inflammatory cytokines, decreased production of the anti-inflammatory and pro-fibrotic cytokine, TGF-beta, and significantly lower levels of collagen deposition, as compared to C57BL/6 control mice. These results demonstrate a role for NADPH oxidase-derived reactive oxygen species in determining course of pulmonary response to SWCNT.

Alteration of deposition pattern and pulmonary response as a result of improved dispersion of aspirated single-walled carbon nanotubes in a mouse model.

Nanoparticles have a fundamental dimension of <100 nm. However, on suspension in media, agglomerates of nanoparticles are the more common structure. This is particularly evident in prior intratracheal instillation or aspiration studies of single-walled carbon nanotubes (SWCNT), in which granulomatous lesions encased by epithelioid macrophages were produced by large agglomerates. In this study, we tested the hypothesis of whether exposure to more dispersed SWCNT structures would alter pulmonary distribution and response. A dispersed preparation of single-walled carbon nanotubes (DSWCNT) with a mean diameter of 0.69 microm was given by pharyngeal aspiration to C57BL/6 mice. Electron microscopy demonstrated a highly dispersed, interstitial distribution of DSWCNT deposits by 1 day postexposure. Deposits were generally <1 microm. Macrophage phagocytosis of DSWCNT was rarely observed at any time point. Lung responses were studied by lavage and morphometry at 1 h, 1 day, 7 day, and 1 mo after a single DSWCNT exposure of 10 microg/mouse. Lung sections and lavage cells demonstrated an early, transient neutrophilic and inflammatory phase that rapidly resolved and was similar to that observed with large agglomerates. No granulomatous lesions or epithelioid macrophages were detected. Morphometric measurement of Sirius red staining was used to assess the connective tissue response. The average thickness of connective tissue in alveolar regions was 0.10 +/- 0.02, 0.09 +/- 0.02, 0.10 +/- 0.01, 0.48 +/- 0.04, and 0.88 +/- 0.19 microm for PBS and 1-h, 1-day, 7-day, and 1-mo postexposure groups, respectively. The results demonstrate that dispersed SWCNT are rapidly incorporated into the alveolar interstitium and that they produce an increase in collagen deposition.