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.

Detection of HOCl-driven degradation of the pericardium scaffolds by label-free multiphoton fluorescence lifetime imaging.

Artificial biomaterials can significantly increase the rate of tissue regeneration. However, implantation of scaffolds leads not only to accelerated tissue healing but also to an immune response of the organism, which results in the degradation of the biomaterial. The synergy of the immune response and scaffold degradation processes largely determines the efficiency of tissue regeneration. Still, methods suitable for fast, accurate and non-invasive characterization of the degradation degree of biomaterial are highly demandable. Here we show the possibility of monitoring the degradation of decellularized bovine pericardium scaffolds under conditions mimicking the immune response and oxidation processes using multiphoton tomography combined with fluorescence lifetime imaging (MPT-FLIM). We found that the fluorescence lifetimes of genipin-induced cross-links in collagen and oxidation products of collagen are prominent markers of oxidative degradation of scaffolds. This was verified in model experiments, where the oxidation was induced with hypochlorous acid or by exposure to activated neutrophils. The fluorescence decay parameters also correlated with the changes of micromechanical properties of the scaffolds as assessed using atomic force microscopy (AFM). Our results suggest that FLIM can be used for quantitative assessments of the properties and degradation of the scaffolds essential for the wound healing processes in vivo.

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.

Interrogating Parkinson's disease associated redox targets: Potential application of CRISPR editing.

Loss of dopaminergic neurons in the substantia nigra is one of the pathogenic hallmarks of Parkinson's disease, yet the underlying molecular mechanisms remain enigmatic. While aberrant redox metabolism strongly associated with iron dysregulation and accumulation of dysfunctional mitochondria is considered as one of the major contributors to neurodegeneration and death of dopaminergic cells, the specific anomalies in the molecular machinery and pathways leading to the PD development and progression have not been identified. The high efficiency and relative simplicity of a new genome editing tool, CRISPR/Cas9, make its applications attractive for deciphering molecular changes driving PD-related impairments of redox metabolism and lipid peroxidation in relation to mishandling of iron, aggregation and oligomerization of alpha-synuclein and mitochondrial injury as well as in mechanisms of mitophagy and programs of regulated cell death (apoptosis and ferroptosis). These insights into the mechanisms of PD pathology may be used for the identification of new targets for therapeutic interventions and innovative approaches to genome editing, including CRISPR/Cas9.

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.

Magic angle spinning 31P NMR spectroscopy reveals two essentially identical ionization states for the cardiolipin phosphates in phospholipid liposomes.

Specific membrane lipid composition is crucial for optimized structural and functional organization of biological membranes. Cardiolipin is a unique phospholipid and important component of the inner mitochondrial membrane. It is involved in energy metabolism, inner mitochondrial membrane transport, regulation of multiple metabolic reactions and apoptotic cell death. The physico-chemical properties of cardiolipin have been studied extensively but despite all these efforts there is still lingering controversy regarding the ionization of the two phosphate groups of cardiolipin. Results obtained in the 1990s and early 2000s suggested that cardiolipin has two disparate pKa values where one of the protons was proposed to be stabilized by an intramolecular hydrogen bond. This has led to extensive speculations on the roles of these two putative ionization states of cardiolipin in mitochondria. More recently the notion of two pKa values has been challenged and rejected by several groups. These studies relied on external measurements of proton adsorption or electrophoretic mobility of membranes but did not take into account the low pH phase behavior and chemical stability of cardiolipin. Here we used 31P NMR to show that in the physiologically relevant membrane phospholipid environment, cardiolipin carries two negative charges at physiological pH. We additionally demonstrate the pH dependent phase behavior and chemical stability of cardiolipin containing membranes.

NDPK-D (NM23-H4)-mediated externalization of cardiolipin enables elimination of depolarized mitochondria by mitophagy.

Mitophagy is critical for cell homeostasis. Externalization of the inner mitochondrial membrane phospholipid, cardiolipin (CL), to the surface of the outer mitochondrial membrane (OMM) was identified as a mitophageal signal recognized by the microtubule-associated protein 1 light chain 3. However, the CL-translocating machinery remains unknown. Here we demonstrate that a hexameric intermembrane space protein, NDPK-D (or NM23-H4), binds CL and facilitates its redistribution to the OMM. We found that mitophagy induced by a protonophoric uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), caused externalization of CL to the surface of mitochondria in murine lung epithelial MLE-12 cells and human cervical adenocarcinoma HeLa cells. RNAi knockdown of endogenous NDPK-D decreased CCCP-induced CL externalization and mitochondrial degradation. A R90D NDPK-D mutant that does not bind CL was inactive in promoting mitophagy. Similarly, rotenone and 6-hydroxydopamine triggered mitophagy in SH-SY5Y cells was also suppressed by knocking down of NDPK-D. In situ proximity ligation assay (PLA) showed that mitophagy-inducing CL-transfer activity of NDPK-D is closely associated with the dynamin-like GTPase OPA1, implicating fission-fusion dynamics in mitophagy regulation.

LC/MS analysis of cardiolipins in substantia nigra and plasma of rotenone-treated rats: Implication for mitochondrial dysfunction in Parkinson's disease.

Exposure to rotenone in vivo results in selective degeneration of dopaminergic neurons and development of neuropathologic features of Parkinson's disease (PD). As rotenone acts as an inhibitor of mitochondrial respiratory complex I, we employed oxidative lipidomics to assess oxidative metabolism of a mitochondria-specific phospholipid, cardiolipin (CL), in substantia nigra (SN) of exposed animals. We found a significant reduction in oxidizable polyunsaturated fatty acid (PUFA)-containing CL molecular species. We further revealed increased contents of mono-oxygenated CL species at late stages of the exposure. Notably, linoleic acid in sn-1 position was the major oxidation substrate yielding its mono-hydroxy- and epoxy-derivatives whereas more readily "oxidizable" fatty acid residues (arachidonic and docosahexaenoic acids) remained non-oxidized. Elevated levels of PUFA CLs were detected in plasma of rats exposed to rotenone. Characterization of oxidatively modified CL molecular species in SN and detection of PUFA-containing CL species in plasma may contribute to better understanding of the PD pathogenesis and lead to the development of new biomarkers of mitochondrial dysfunction associated with this disease.

Imaging mass spectrometry of diversified cardiolipin molecular species in the brain.

MALDI imaging mass spectrometry (MALDI-IMS) has been used successfully in mapping different lipids in tissue sections, yet existing protocols fail to detect the diverse species of mitochondria-unique cardiolipins (CLs) in the brain which are essential for cellular and mitochondrial physiology. We have developed methods enabling the imaging of individual CLs in brain tissue. This was achieved by eliminating ion suppressive effects by (i) cross-linking carboxyl/amino containing molecules on tissue with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride and (ii) removing highly abundant phosphatidylcholine head groups via phospholipase C treatment. These treatments allowed the detection of CL species at 100 µm resolution and did not affect the amount or molecular species distribution of brain tissue CLs. When combined with augmented matrix application, these modifications allowed the visualization and mapping of multiple CL species in various regions of the brain including the thalamus, hippocampus, and cortex. Areas such as the dentate and stratum radiatum exhibited higher CL signals than other areas within the hippocampal formation. The habenular nuclear (Hb)/dorsal third ventricle (D3 V) and lateral ventricle (LV) areas were identified as CL "hot spots". Our method also allowed structural MS/MS fragmentation and mapping of CLs with identified fatty acid residues and demonstrated a nonrandom distribution of individual oxidizable (polyunsaturated fatty acid containing) and nonoxidizable (nonpolyunsaturated containing) CLs in different anatomical areas of the brain. To our knowledge, this method is the first label-free approach for molecular mapping of diversified CLs in brain tissue.

Improved spatial resolution of matrix-assisted laser desorption/ionization imaging of lipids in the brain by alkylated derivatives of 2,5-dihydroxybenzoic acid.

RATIONALE: Matrix-assisted laser desorption/ionization (MALDI) is one of the major techniques for mass spectrometry imaging (MSI) of biological systems along with secondary-ion mass spectrometry (SIMS) and desorption electrospray mass spectrometry (DESI). The inherent variability of MALDI-MSI signals within intact tissues is related to the heterogeneity of both the sample surface and the matrix crystallization. To circumvent some of these limitations of MALDI-MSI, we have developed improved matrices for lipid analysis based on structural modification of the commonly used matrix 2,5-dihydroxybenzoic acid (DHB). METHODS: We have synthesized DHB containing -C6H13 and -C12H25 alkyl chains and applied these matrices to rat brain using a capillary sprayer. We utilized a Bruker Ultraflex II MALDI-TOF/TOF mass spectrometer to analyze lipid extracts and tissue sections, and examined these sections with polarized light microscopy and differential interference contrast microscopy. RESULTS: O-alkylation of DHB yields matrices, which, when applied to brain sections, follow a trend of phase transition from crystals to an oily layer in the sequence DHB → DHB-C6H13 → DHB-C12H25 . MALDI-MSI images acquired with DHB-C12H25 exhibited a considerably higher density of lipids than DHB. CONCLUSIONS: Comparative experiments with DHB and DHB-C12H25 are presented, which indicate that the latter matrix affords higher lateral resolution than the former.

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic 'eat-me' signals: cleavage and inhibition of phagocytosis by Lp-PLA2.

Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H2O2 was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A2 (Lp-PLA2), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA2 was blocked by a selective inhibitor of Lp-PLA2, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic 'eat-me' signal that participates in the engulfment of cells undergoing intrinsic apoptosis.

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.

Dihydroquercetin (taxifolin) and other flavonoids as inhibitors of free radical formation at key stages of apoptosis.

Formation of free radicals in mitochondria plays a key role in the development of apoptosis, which includes formation of superoxide by the respiratory chain, formation of radicals by cytochrome c-cardiolipin complex in the presence of hydrogen peroxide or lipids, and chain lipid peroxidation resulting in cytochrome c release from mitochondria and initiation of the apoptotic cascade. In this work the effect of taxifolin (dihydroquercetin) and some other antioxidants on these three radical-producing reactions was studied. Peroxidase activity of the complex of cytochrome c with dioleyl cardiolipin estimated by chemiluminescence with luminol decreased by 50% with quercetin, taxifolin, rutin, Trolox, and ionol at concentrations 0.7, 0.7, 0.8, 3, and 10 microM, respectively. The lipid radical production detected by coumarin C-525-activated chemiluminescence decreased under the action of rutin and taxifolin in a dose-dependent manner, so that a 50% inhibition of chemiluminescence was observed at the antioxidant concentrations of 3.7 and 10 microM, respectively. Thus, these two radical-producing reactions responsible for apoptosis onset are inhibited by antioxidants at rather low concentrations. Experiments performed on liver slices and mash showed that taxifolin, quercetin, naringenin, and Trolox have low inhibitory effect on the lucigenin-dependent chemiluminescence in the tissue only at concentrations higher than 100 microM.

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.

Molecular design of new inhibitors of peroxidase activity of cytochrome c/cardiolipin complexes: fluorescent oxadiazole-derivatized cardiolipin.

Interaction of a mitochondria-specific anionic phospholipid, cardiolipin (CL), with an intermembrane protein, cytochrome c (cyt c), yields a peroxidase complex. During apoptosis, the complex induces accumulation of CL oxidation products that are essential for detachment of cyt c from the mitochondrial membrane, induction of permeability transition, and release of proapoptotic factors into the cytosol. Therefore, suppression of the peroxidase activity and prevention of CL oxidation may lead to discovery of new antiapoptotic drugs. Here, we report a new approach to regulate the cyt c peroxidase activity by using modified CL with an oxidizable and fluorescent 7-nitro-2,1,3-benzoxadiazole (NBD) moiety (NBD-CL). We demonstrate that NBD-CL forms high-affinity complexes with cyt c and blocks cyt c-catalyzed oxidation of several peroxidase substrates, cyt c self-oxidation, and, most importantly, inhibits cyt c-dependent oxidation of polyunsaturated tetralinoleoyl CL (TLCL) and accumulation of TLCL hydroperoxides. Electrospray ionization mass spectrometry and fluorescence analysis revealed that oxidation and cleavage of the NBD moiety of NBD-CL underlie the inhibition mechanism. We conclude that modified CL combining a nonoxidizable monounsaturated trioleoyl CL with a C(12)-NBD fragment undergoes a regiospecific oxidation thereby representing a novel inhibitor of cyt c peroxidase activity.

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.