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.

A huge diversification of phospholipids, forming the aqueous interfaces of all biomembranes, cannot be accommodated within a simple concept of their role as membrane building blocks. Indeed, a number of signaling functions of (phospho)lipid molecules has been discovered. Among these signaling lipids, a particular group of oxygenated polyunsaturated fatty acids (PUFA), so called lipid mediators, has been thoroughly investigated over several decades. This group includes oxygenated octadecanoids, eicosanoids, and docosanoids and includes several hundreds of individual species. Oxygenation of PUFA can occur when they are esterified into major classes of phospholipids. Initially, these events have been associated with non-specific oxidative injury of biomembranes. An alternative concept is that these post-synthetically oxidatively modified phospholipids and their adducts with proteins are a part of a redox epiphospholipidome that represents a rich and versatile language for intra- and inter-cellular communications. The redox epiphospholipidome may include hundreds of thousands of individual molecular species acting as meaningful biological signals. This review describes the signaling role of oxygenated phospholipids in programs of regulated cell death. Although phospholipid peroxidation has been associated with almost all known cell death programs, we chose to discuss enzymatic pathways activated during apoptosis and ferroptosis and leading to peroxidation of two phospholipid classes, cardiolipins (CLs) and phosphatidylethanolamines (PEs). This is based on the available LC-MS identification and quantitative information on the respective peroxidation products of CLs and PEs. We focused on molecular mechanisms through which two proteins, a mitochondrial hemoprotein cytochrome c (cyt c), and non-heme Fe lipoxygenase (LOX), change their catalytic properties to fulfill new functions of generating oxygenated CL and PE species. Given the high selectivity and specificity of CL and PE peroxidation we argue that enzymatic reactions catalyzed by cyt c/CL complexes and 15-lipoxygenase/phosphatidylethanolamine binding protein 1 (15LOX/PEBP1) complexes dominate, at least during the initiation stage of peroxidation, in apoptosis and ferroptosis. We contrast cell-autonomous nature of CLox signaling in apoptosis correlating with its anti-inflammatory functions vs. non-cell-autonomous ferroptotic signaling facilitating pro-inflammatory (necro-inflammatory) responses. Finally, we propose that small molecule mechanism-based regulators of enzymatic phospholipid peroxidation may lead to highly specific anti-apoptotic and anti-ferroptotic therapeutic modalities.

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.

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.

In addition to the known prominent role of polyunsaturated (phospho)lipids as structural blocks of biomembranes, there is an emerging understanding of another important function of these molecules as a highly diversified signaling language utilized for intra- and extracellular communications. Technological developments in high-resolution mass spectrometry facilitated the development of a new branch of metabolomics, redox lipidomics. Analysis of lipid peroxidation reactions has already identified specific enzymatic mechanisms responsible for the biosynthesis of several unique signals in response to inflammation and regulated cell death programs. Obtaining comprehensive information about millions of signals encoded by oxidized phospholipids, represented by thousands of interactive reactions and pleiotropic (patho)physiological effects, is a daunting task. However, there is still reasonable hope that significant discoveries, of at least some of the important contributors to the overall overwhelmingly complex network of interactions triggered by inflammation, will lead to the discovery of new small molecule regulators and therapeutic modalities. For example, suppression of the production of AA-derived pro-inflammatory mediators, HXA3 and LTB4, by an iPLA2 gamma inhibitor, R-BEL, mitigated injury associated with the activation of pro-inflammatory processes in animals exposed to whole-body irradiation. Further, technological developments promise to make redox lipidomics a powerful approach in the arsenal of diagnostic and therapeutic instruments for personalized medicine of inflammatory diseases and conditions.

SIGNIFICANCE: Oxygenated polyunsaturated lipids are known to play multi-functional roles as essential signals coordinating metabolism and physiology. Among them are well-studied eicosanoids and docosanoids that are generated via phospholipase A2 hydrolysis of membrane phospholipids and subsequent oxygenation of free polyunsaturated fatty acids (PUFA) by cyclooxygenases and lipoxygenases. Recent Advances: There is an emerging understanding that oxygenated PUFA-phospholipids also represent a rich signaling language with yet-to-be-deciphered details of the execution machinery-oxygenating enzymes, regulators, and receptors. Both free and esterified oxygenated PUFA signals are generated in cells, and their cross-talk and inter-conversion through the de-acylation/re-acylation reactions is not sufficiently explored. CRITICAL ISSUES: Here, we review recent data related to oxygenated phospholipids as important damage signals that trigger programmed cell death pathways to eliminate irreparably injured cells and preserve the health of multicellular environments. We discuss the mechanisms underlying the trans-membrane redistribution and generation of oxygenated cardiolipins in mitochondria by cytochrome c as pro-apoptotic signals. We also consider the role of oxygenated phosphatidylethanolamines as proximate pro-ferroptotic signals. FUTURE DIRECTIONS: We highlight the importance of sequential processes of phospholipid oxygenation and signaling in disease contexts as opportunities to use their regulatory mechanisms for the identification of new therapeutic targets.

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.

The interaction between CL and cytochrome c (cyt-c), results in a gain of function of peroxidase activity by cyt-c. Despite intensive research, disagreements on nature and molecular details of this interaction remain. In particular, it is still not known how the interaction triggers the onset of apoptosis. Enzymatic characterization of peroxidase activity has highlighted the need for a critical threshold concentration of CL, a finding of profound physiological relevance in vivo. Using solution NMR, fluorescence spectroscopy, and in silico modeling approaches we here confirm that full binding of cyt-c to the membrane requires a CL:cyt-c threshold ratio of 5:1. Among three binding sites, the simultaneous binding of two sites, at two opposing sides of the heme, provides a mechanism to open the heme crevice to substrates, resulting in "productive binding" in which cyt-c then sequesters CL, inducing curvature in the membrane. Membrane perturbation along with lipid peroxidation, due to interactions of heme/CL acyl chains, initiates the next step in the apoptotic pathway of making the membrane leaky. The third CL binding site while allowing interaction with the membrane, does not cluster CL or induce subsequent events, making this interaction "unproductive".

PURPOSE OF THE STUDY: A number of in vivo studies have shown that pulmonary exposure to carbon nanotubes (CNTs) may lead to an acute local inflammatory response, pulmonary fibrosis, and granulomatous lesions. Among the factors that play direct roles in initiation and progression of fibrotic processes are epithelial-mesenchymal transition and myofibroblasts recruitment/differentiation, both mediated by transforming growth factor-beta1 (TGF-beta1). Yet, other contributors to TGF-beta1 associated signaling, such as osteopontin (OPN) has not been fully investigated. MATERIALS AND METHODS: OPN-knockout female mice (OPN-KO) along with their wild-type (WT) counterparts were exposed to single-walled carbon nanotubes (SWCNT) (40 microg/mouse) via pharyngeal aspiration and fibrotic response was assessed 1, 7, and 28 days post-exposure. Simultaneously, RAW 264.7 and MLE-15 cells were treated with SWCNT (24 hours, 6 microg/cm(2) to 48 microg/cm(2)) or bleomycin (0.1 microg/ml) in the presence of OPN-blocking antibody or isotype control, and TGF-beta1 was measured in supernatants. RESULTS AND CONCLUSIONS: Diminished lactate dehydrogenase activity at all time points, along with less pronounced neutrophil influx 24 h post-exposure, were measured in broncho-alveolar lavage (BAL) of OPN-KO mice compared to WT. Pro-inflammatory cytokine release (IL-6, TNF-alpha, MCP-1) was reduced. A significant two-fold increase of TGF-beta1 was found in BAL of WT mice at 7 days, while TGF-beta1 levels in OPN-KO animals remained unaltered. Histological examination revealed marked decrease in granuloma formation and less collagen deposition in the lungs of OPN-KO mice compared to WT. RAW 264.7 but not MLE-15 cells exposed to SWCNT and bleomycin had significantly less TGF-beta1 released in the presence of OPN-blocking antibody. We believe that OPN is important in initiating the cellular mechanisms that produce an overall pathological response to SWCNT and it may act upstream of TGF-beta1. Further investigation to understand the mechanistic details of such interactions is critical to predict outcomes of pulmonary exposure to CNT.

According to the Centers for Disease Control and Prevention, approximately 25 million Americans suffer from asthma. The disease total annual cost is about $56 billion and includes both the direct and indirect costs of medications, hospital stays, missed work, and decreased productivity. Air pollution with xenobiotics, bacterial agents, and industrial nanomaterials, such as carbon nanotubes, contribute to the exacerbation of this condition and are a point of particular attention in environmental toxicology as well as in occupational health and safety research. Mast cell degranulation and activation of Th2 cells triggered either by allergen-specific immunoglobulin E (IgE) or by alternative mechanisms, such as locally produced neurotransmitters, underlie the pathophysiological process of airway constriction during an asthma attack. Other immune and non-immune cell types, including basophils, eosinophils, Th1, Th17, Th9, macrophages, dendritic cells, and smooth muscle cells, are involved in the inflammatory and allergic responses during asthma, which, under chronic conditions, may progress without mast cells, the key trigger of the acute asthma attack. To decipher complex molecular, cellular, and genetic mechanisms, many researchers have attempted to develop in vitro and in vivo models to study asthma. Herein, we summarize the advantages and disadvantages of various models and their applicability to nanoparticle evaluation in asthma research. We further suggest that a framework for both in vitro and in vivo methods should be used to study the impact of engineered nanomaterials on asthma etiology, pathophysiology, and treatment.

BACKGROUND: Cellulose-based materials have been used for centuries to manufacture different goods derived from forestry and agricultural sources. In the growing field of nanocellulose applications, its uniquely engineered properties are instrumental for inventive products coming to competitive markets. Due to their high aspect ratio and stiffness, it is speculated that cellulose nanocrystals (CNC) may cause similar pulmonary toxicity as carbon nanotubes and asbestos, thus posing a potential negative impact on public health and the environment. METHODS: The present study was undertaken to investigate the pulmonary outcomes induced by repeated exposure to respirable CNC. C57BL/6 female and male mice were exposed by pharyngeal aspiration to CNC (40 mug/mouse) 2 times a week for 3 weeks. Several biochemical endpoints and pathophysiological outcomes along with gene expression changes were evaluated and compared in the lungs of male and female mice. RESULTS: Exposure to respirable CNC caused pulmonary inflammation and damage, induced oxidative stress, elevated TGF-beta and collagen levels in lung, and impaired pulmonary functions. Notably, these effects were markedly more pronounced in females compared to male mice. Moreover, sex differences in responses to pulmonary exposure to CNC were also detected at the level of global mRNA expression as well as in inflammatory cytokine/chemokine activity. CONCLUSIONS: Overall, our results indicate that there are considerable differences in responses to respirable CNC based on gender with a higher pulmonary toxicity observed in female mice.

Biopersistence of carbon nanotubes, graphene oxide (GO) and several other types of carbonaceous nanomaterials is an essential determinant of their health effects. Successful biodegradation is one of the major factors defining the life span and biological responses to nanoparticles. Here, we review the role and contribution of different oxidative enzymes of inflammatory cells - myeloperoxidase, eosinophil peroxidase, lactoperoxidase, hemoglobin, and xanthine oxidase - to the reactions of nanoparticle biodegradation. We further focus on interactions of nanomaterials with hemoproteins dependent on the specific features of their physico-chemical and structural characteristics. Mechanistically, we highlight the significance of immobilized peroxidase reactive intermediates vs diffusible small molecule oxidants (hypochlorous and hypobromous acids) for the overall oxidative biodegradation process in neutrophils and eosinophils. We also accentuate the importance of peroxynitrite-driven pathways realized in macrophages via the engagement of NADPH oxidase- and NO synthase-triggered oxidative mechanisms. We consider possible involvement of oxidative machinery of other professional phagocytes such as microglial cells, myeloid-derived suppressor cells, in the context of biodegradation relevant to targeted drug delivery. We evaluate the importance of genetic factors and their manipulations for the enzymatic biodegradation in vivo. Finally, we emphasize a novel type of biodegradation realized via the activation of the "dormant" peroxidase activity of hemoproteins by the nano-surface. This is exemplified by the binding of GO to cyt c causing the unfolding and 'unmasking' of the peroxidase activity of the latter. We conclude with the strategies leading to safe by design carbonaceous nanoparticles with optimized characteristics for mechanism-based targeted delivery and regulatable life-span of drugs in circulation.

During last decades, changes have been observed in the frequency of different histological subtypes of lung cancer - one of the most common causes of morbidity and mortality - with a declining proportion of squamous cell carcinomas and an increasing proportion of adenocarcinomas, particularly in developed countries. This suggests the emergence of new etiological factors and mechanisms including those defining the lung microenvironment promoting tumor growth. Assuming that the lung is the main portal of entry for broadly used nanomaterials and their established pro-inflammatory propensities, we hypothesized that nanomaterials may contribute to changes facilitating tumor growth. Here we report that an acute exposure to single-walled carbon nanotubes (SWCNT) induces recruitment and accumulation of lung-associated myeloid-derived suppressor cells (MDSC) and MDSC-derived production of TGF-beta resulting in up-regulated tumor burden in the lung. The production of TGF-beta by MDSC requires their interaction with both SWCNT and tumor cells. We conclude that pulmonary exposure to SWCNT favors the formation of a niche that supports ingrowth of lung carcinoma in vivo via activation of TGF-beta production by SWCNT-attracted and pre-sensitized MDSC.

Altering the fuel source from petroleum-based ultralow sulfur diesel to biodiesel and its blends is considered by many to be a sustainable choice for controlling exposures to particulate material. As the exhaust of biodiesel/diesel blends is composed of a combination of combustion products of polycyclic aromatic hydrocarbons and fatty acid methyl esters, we hypothesize that 50% biodiesel/diesel blend (BD50) exposure could induce harmful outcomes because of its ability to trigger oxidative damage. Here, adverse effects were compared in murine male reproductive organs after pharyngeal aspiration with particles generated by engine fueled with BD50 or neat petroleum diesel (D100). When compared with D100, exposure to BD50 significantly altered sperm integrity, including concentration, motility, and morphological abnormalities, as well as increasing testosterone levels in testes during the time course postexposure. Serum level of luteinizing hormone was significantly depleted only after BD50 exposure. Moreover, we observed that exposure to BD50 significantly increased sperm DNA fragmentation and the upregulation of inflammatory cytokines in the serum and testes on Day 7 postexposure when compared with D100. Histological evaluation of testes sections from BD50 exposure indicated more noticeable interstitial edema, degenerating spermatocytes, and dystrophic seminiferous tubules with arrested spermatogenesis. Significant differences in the level of oxidative stress assessed by accumulation of lipid peroxidation products and depletion of glutathione were detected on exposure to respirable BD50 and D100. Taken together, these results indicate that exposure of mice to inhalable BD50 caused more pronounced adverse effects on male reproductive function than diesel.

The use of cellulose as building blocks for the development of novel functional materials is rapidly growing. Cellulose nanocrystals (CNC), with advantageous chemical and mechanical properties, have gained prominence in a number of applications, such as in nanofillers in polymer composites, building materials, cosmetics, food, and the drug industry. Therefore, it becomes critical to evaluate the potential health effects associated with CNC exposures. The objective of this study was to compare pulmonary outcomes caused by exposure of C57BL/6 mice to two different processed forms of CNC derived from wood, i.e., CNCS (10 wt %; gel/suspension) and CNCP (powder), and compare to asbestos induced responses. Pharyngeal aspiration with CNCS and CNCP was found to facilitate innate inflammatory response assessed by an increase in leukocytes and eosinophils recovered by bronchoalveolar lavage (BAL). Biomarkers of tissue damage were elevated to a higher extent in mice exposed to CNCP. Compared to CNCP, CNCS caused a significant increase in the accumulation of oxidatively modified proteins. The up-regulation of inflammatory cytokines was higher in the lungs after CNCS treatments. Most importantly, CNCP materials were significantly longer than CNCS. Taken together, our data suggests that particle morphology and nanosize dimensions of CNCs, regardless of the same source, may be critical factors affecting the type of innate immune inflammatory responses. Because various processes have been developed for producing highly sophisticated nanocellulose materials, detailed assessment of specific health outcomes with respect to their physical-structural-chemical properties is highly warranted.

In contrast to short-lived neutrophils, macrophages display persistent presence in the lung of animals after pulmonary exposure to carbon nanotubes. While effective in the clearance of bacterial pathogens and injured host cells, the ability of macrophages to "digest" carbonaceous nanoparticles has not been documented. Here, we used chemical, biochemical, and cell and animal models and demonstrated oxidative biodegradation of oxidatively functionalized single-walled carbon nanotubes via superoxide/NO* --> peroxynitrite-driven oxidative pathways of activated macrophages facilitating clearance of nanoparticles from the lung.

Several lines of evidence indicate that exposure to nanoparticles (NPs) is able to modify airway immune responses, thus facilitating the development of respiratory diseases. Graphene oxide (GO) is a promising carbonaceous nanomaterial with unique physicochemical properties, envisioned for a multitude of medical and industrial applications. In this paper, we determined how exposure to GO modulates the allergic pulmonary response. Using a murine model of ovalbumin (OVA)-induced asthma, we revealed that GO, given at the sensitization stage, augmented airway hyperresponsiveness and airway remodeling in the form of goblet cell hyperplasia and smooth muscle hypertrophy. At the same time, the levels of the cytokines IL-4, IL-5, and IL-13 were reduced in broncho-alveolar lavage (BAL) fluid in GO-exposed mice. Exposure to GO during sensitization with OVA decreased eosinophil accumulation and increased recruitment of macrophages in BAL fluid. In line with the cytokine profiles, sensitization with OVA in the presence of GO stimulated the production of OVA-specific IgG2a and down-regulated the levels of IgE and IgG1. Moreover, exposure to GO increased the macrophage production of the mammalian chitinases, CHI3L1 and AMCase, whose expression is associated with asthma. Finally, molecular modeling has suggested that GO may directly interact with chitinase, affecting AMCase activity, which has been directly proven in our studies. Thus, these data show that GO exposure attenuates Th2 immune response in a model of OVA-induced asthma, but leads to potentiation of airway remodeling and hyperresponsiveness, with the induction of mammalian chitinases.

Eosinophil peroxidase (EPO) is one of the major oxidant-producing enzymes during inflammatory states in the human lung. The degradation of single-walled carbon nanotubes (SWCNTs) upon incubation with human EPO and H2 O2 is reported. Biodegradation of SWCNTs is higher in the presence of NaBr, but neither EPO alone nor H2 O2 alone caused the degradation of nanotubes. Molecular modeling reveals two binding sites for SWCNTs on EPO, one located at the proximal side (same side as the catalytic site) and the other on the distal side of EPO. The oxidized groups on SWCNTs in both cases are stabilized by electrostatic interactions with positively charged residues. Biodegradation of SWCNTs can also be executed in an ex vivo culture system using primary murine eosinophils stimulated to undergo degranulation. Biodegradation is proven by a range of methods including transmission electron microscopy, UV-visible-NIR spectroscopy, Raman spectroscopy, and confocal Raman imaging. Thus, human EPO (in vitro) and ex vivo activated eosinophils mediate biodegradation of SWCNTs: an observation that is relevant to pulmonary responses to these materials.

The use of biodiesel (BD) or its blends with petroleum diesel (D) is considered to be a viable approach to reduce occupational and environmental exposures to particulate matter (PM). Due to its lower particulate mass emissions compared to D, use of BD is thought to alleviate adverse health effects. Considering BD fuel is mainly composed of unsaturated fatty acids, we hypothesize that BD exhaust particles could induce pronounced adverse outcomes, due to their ability to readily oxidize. The main objective of this study was to compare the effects of particles generated by engine fueled with neat BD and neat petroleum-based D. Biomarkers of tissue damage and inflammation were significantly elevated in lungs of mice exposed to BD particulates. Additionally, BD particulates caused a significant accumulation of oxidatively modified proteins and an increase in 4-hydroxynonenal. The up-regulation of inflammatory cytokines/chemokines/growth factors was higher in lungs upon BD particulate exposure. Histological evaluation of lung sections indicated presence of lymphocytic infiltrate and impaired clearance with prolonged retention of BD particulate in pigment laden macrophages. Taken together, these results clearly indicate that BD exhaust particles could exert more toxic effects compared to D.

Metastatic establishment and growth of Lewis lung carcinoma is promoted by single-walled carbon nanotubes (SWCNT) in C57BL6/J mice. The effect is mediated by increased local and systemic accumulation of myeloid-derived suppressor cells (MDSC), as their depletion abrogated pro-tumor activity in vivo. These data are important for the design of novel theranostics platforms with modules capable of depleting or functionally suppressing MDSC to ensure effective immunosurveillance in the tumor microenvironment. (Copyright 2013 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim.)

Graphene oxide (GO) and C60- or C60-TRIS fullerenes, internalized by murine dendritic cells (DCs), differently affect their abilities to present antigens to T-cells. While C60-fullerenes stimulate the ovalbumin-specific MHC class I-restricted T-cell response, GO impairs the stimulatory potential of DCs. In contrast to C60-fullerenes, GO decreases the intracellular levels of LMP7 immunoproteasome subunits required for processing of protein antigens. This is important for the development of DC-based vaccines. (Copyright 2013 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim.)

Over the past two decades, nanotechnology has emerged as a key player in various disciplines of science and technology. Some of the most exciting applications are in the field of biomedicine - for theranostics (for combined diagnostic and therapeutic purposes) as well as for exploration of biological systems. A detailed understanding of the molecular details of interactions between nanoparticles and biological nano-machinery - macromolecules, membranes, and intracellular organelles - is crucial for obtaining adequate information on mechanisms of action of nanomaterials as well as a perspective on the long term effects of these materials and their possible toxicological outcomes. This review focuses on the use of structure-based computational molecular modeling as a tool to understand and to predict the interactions between nanomaterials and nano-biosystems. We review major approaches and provide examples of computational analysis of the structural principles behind such interactions. A rationale on how nanoparticles of different sizes, shape, structure and chemical properties can affect the organization and functions of nano-machinery of cells is also presented.

Over the past three decades, revolutionary research in nanotechnology by the scientific, medical, and engineering communities has yielded a treasure trove of discoveries with diverse applications that promise to benefit humanity. With their unique electronic and mechanical properties, carbon nanomaterials (CNMs) represent a prime example of the promise of nanotechnology with applications in areas that include electronics, fuel cells, composites, and nanomedicine. Because of toxicological issues associated with CNMs, however, their full commercial potential may not be achieved. The ex vitro, in vitro, and in vivo data presented in this Account provide fundamental insights into the biopersistence of CNMs, such as carbon nanotubes and graphene, and their oxidation/biodegradation processes as catalyzed by peroxidase enzymes. We also communicate our current understanding of the mechanism for the enzymatic oxidation and biodegradation. Finally, we outline potential future directions that could enhance our mechanistic understanding of the CNM oxidation and biodegradation and could yield benefits in terms of human health and environmental safety. The conclusions presented in this Account may catalyze a rational rethinking of CNM incorporation in diverse applications. For example, armed with an understanding of how and why CNMs undergo enzyme-catalyzed oxidation and biodegradation, researchers can tailor the structure of CNMs to either promote or inhibit these processes. In nanomedical applications such as drug delivery, the incorporation of carboxylate functional groups could facilitate biodegradation of the nanomaterial after delivery of the cargo. On the other hand, in the construction of aircraft, a CNM composite should be stable to oxidizing conditions in the environment. Therefore, pristine, inert CNMs would be ideal for this application. Finally, the incorporation of CNMs with defect sites in consumer goods could provide a facile mechanism that promotes the degradation of these materials once these products reach landfills.

Oxidized phospholipid species are important, biologically relevant, lipid signaling molecules that usually exist in low abundance in biological tissues. Along with their inherent stability issues, these oxidized lipids present themselves as a challenge in their detection and identification. Often times, oxidized lipid species can co-chromatograph with non-oxidized species making the detection of the former extremely difficult, even with the use of mass spectrometry. In this study, a normal-phase and reverse-phase two dimensional high performance liquid chromatography (HPLC)-mass spectrometric system was applied to separate oxidized phospholipids from their non-oxidized counterparts, allowing unambiguous detection in a total lipid extract. We have utilized bovine heart cardiolipin as well as commercially available tetralinoleoyl cardiolipin oxidized with cytochrome c (cyt c) and hydrogen peroxide as well as with lipoxygenase to test the separation power of the system. Our findings indicate that oxidized species of not only cardiolipin, but other phospholipid species, can be effectively separated from their non-oxidized counterparts in this two dimensional system. We utilized three types of biological tissues and oxidative insults, namely rotenone treatment of lymphocytes to induce mitochondrial damage and cell death, pulmonary inhalation exposure to single walled carbon nanotubes, as well as total body irradiation, in order to identify cardiolipin oxidation products, critical to the cell damage/cell death pathways in these tissues following cellular stress/injury. Our results indicate that selective cardiolipin (CL) oxidation is a result of a non-random free radical process. In addition, we assessed the ability of the system to identify CL oxidation products in the brain, a tissue known for its extreme complexity and diversity of CL species. The ability of the two dimensional HPLC-mass spectrometric system to detect and characterize oxidized lipid products will allow new studies to be formulated to probe the answers to biologically important questions with regard to oxidative lipidomics and cellular insult. This article is part of a Special Issue entitled: Oxidized phospholipids - their properties and interactions with proteins.

A number of commercially available metal/metal oxide nanoparticles (NPs) such as superparamagnetic iron oxide (SPION) are utilized by the medical field for a wide variety of applications. These NPs may able to induce dermal toxicity via their physical nature and reactive surface properties. We hypothesize that SPION may be toxic to skin via the ability of particles to be internalized and thereby initiate oxidative stress, inducing redox-sensitive transcription factors affecting/leading to inflammation. Due to the skin's susceptibility to UV radiation, it is also of importance to address the combined effect of UVB and NPs co-exposure. To test this hypothesis, the effects of dextran-coated SPION of different sizes (15-50 nm) and manufacturers (MicroMod, Rostock-Warnemunde, Germany and KTH-Royal Institute of Technology, Stockholm, Sweden) were evaluated in two cell lines: normal human epidermal keratinocytes (HEK) and murine epidermal cells (JB6 P(+)). HEK cells exposed to 20 nm (KTH and MicroMod) had a decrease in viability, while the 15 and 50 nm particles were not cytotoxic. HEK cells were also capable of internalizing the KTH particles (15 and 20 nm) but not the MicroMod SPION (20 and 50 nm). IL-8 and IL-6 were also elevated in HEK cells following exposure to SPION. Exposure of JB6 P(+) cells to all SPIONs evaluated resulted in activation of AP-1. Exposure to SPION alone was not sufficient to induce NF-kappaB activation; however, co-exposure with UVB resulted in significant NF-kappaB induction in cells exposed to 15 and 20 nm KTH SPION and 50 nm MicroMod particles. Pre-exposure of JB6 P(+) cells to UVB followed by NPs induced a significant depletion of glutathione, release of cytokines, and cell damage as assessed by release of lactate dehydrogenase. Altogether, these data indicate that co-exposure to UVB and SPIONs was associated with induction of oxidative stress and release of inflammatory mediators. These results verify the need to thoroughly evaluate the adverse effects of UVB when evaluating dermal toxicity of engineered NPs on skin.

The pulmonary route represents one of the most important portals of entry for nanoparticles into the body. However, the in vivo interactions of nanoparticles with biomolecules of the lung have not been sufficiently studied. Here, using an established mouse model of pharyngeal aspiration of single-walled carbon nanotubes (SWCNTs), we recovered SWCNTs from the bronchoalveolar lavage fluid (BALf), purified them from possible contamination with lung cells, and examined the composition of phospholipids adsorbed on SWCNTs by liquid chromatography mass spectrometry (LC-MS) analysis. We found that SWCNTs selectively adsorbed two types of the most abundant surfactant phospholipids: phosphatidylcholines (PC) and phosphatidylglycerols (PG). Molecular speciation of these phospholipids was also consistent with pulmonary surfactant. Quantitation of adsorbed lipids by LC-MS along with the structural assessments of phospholipid binding by atomic force microscopy and molecular modeling indicated that the phospholipids ( approximately 108 molecules per SWCNT) formed an uninterrupted "coating" whereby the hydrophobic alkyl chains of the phospholipids were adsorbed onto the SWCNT with the polar head groups pointed away from the SWCNT into the aqueous phase. In addition, the presence of surfactant proteins A, B, and D on SWCNTs was determined by LC-MS. Finally, we demonstrated that the presence of this surfactant coating markedly enhanced the in vitro uptake of SWCNTs by macrophages. Taken together, this is the first demonstration of the in vivo adsorption of the surfactant lipids and proteins on SWCNTs in a physiologically relevant animal model.

Nanotechnologies are emerging as highly promising technologies in many sectors in the society. However, the increasing use of engineered nanomaterials also raises concerns about inadvertent exposure to these materials and the potential for adverse effects on human health and the environment. Despite several years of intensive investigations, a common paradigm for the understanding of nanoparticle-induced toxicity remains to be firmly established. Here, the so-called oxidative stress paradigm is scrutinized. Does oxidative stress represent a secondary event resulting inevitably from disruption of biochemical processes and the demise of the cell, or a specific, non-random event that plays a role in the induction of cellular damage e.g. apoptosis? The answer to this question will have important ramifications for the development of strategies for mitigation of adverse effects of nanoparticles. Recent examples of global lipidomics studies of nanoparticle-induced tissue damage are discussed along with proteomics and transcriptomics approaches to achieve a comprehensive understanding of the complex and interrelated molecular changes in cells and tissues exposed to nanoparticles. We also discuss instances of non-oxidative stress-mediated cellular damage resulting from direct physical interference of nanomaterials with cellular structures.

Advancement of biomedical applications of carbonaceous nanomaterials is hampered by their biopersistence and pro-inflammatory action in vivo. Here, we used myeloperoxidase knockout B6.129X1-MPO (MPO k/o) mice and showed that oxidation and clearance of single walled carbon nanotubes (SWCNT) from the lungs of these animals after pharyngeal aspiration was markedly less effective whereas the inflammatory response was more robust than in wild-type C57Bl/6 mice. Our results provide direct evidence for the participation of MPO - one of the key-orchestrators of inflammatory response - in the in vivo pulmonary oxidative biodegradation of SWCNT and suggest new ways to control the biopersistence of nanomaterials through genetic or pharmacological manipulations.

It is commonly believed that nanomaterials cause nonspecific oxidative damage. Our mass spectrometry-based oxidative lipidomics analysis of all major phospholipid classes revealed highly selective patterns of pulmonary peroxidation after inhalation exposure of mice to single-walled carbon nanotubes. No oxidized molecular species were found in the two most abundant phospholipid classes: phosphatidylcholine and phosphatidylethanolamine. Peroxidation products were identified in three relatively minor classes of anionic phospholipids, cardiolipin, phosphatidylserine, and phosphatidylinositol, whereby oxygenation of polyunsaturated fatty acid residues also showed unusual substrate specificity. This nonrandom peroxidation coincided with the accumulation of apoptotic cells in the lung. A similar selective phospholipid peroxidation profile was detected upon incubation of a mixture of total lung lipids with H(2)O(2)/cytochrome c known to catalyze cardiolipin and phosphatidylserine peroxidation in apoptotic cells. The characterized specific phospholipid peroxidation signaling pathways indicate new approaches to the development of mitochondria-targeted regulators of cardiolipin peroxidation to protect against deleterious effects of pro-apoptotic effects of single-walled carbon nanotubes in the lung.

Pharyngeal aspiration of single-walled carbon nanotubes (SWCNTs) caused inflammation, pulmonary damage, and an altered cytokine network in the lung. Local inflammatory response in vivo was accompanied by modified systemic immunity as documented by decreased proliferation of splenic T cells. Preincubation of naive T cells in vitro with SWCNT-treated dendritic cells reduced proliferation of T cells. Our data suggest that in vivo exposure to SWCNT modifies systemic immunity by modulating dendritic cell function.