Day 1: October 28, 2009

SESSION I: Inflammation, Lung Cancer and Fibroproliferative Diseases: Roles of Reactive Oxygen/Nitrogen Species

Reactive Oxygen/Nitrogen Species in Lung Cancer

Neelam Azad, PhD¹, Anand Iyer, PhD¹, Val Vallyathan, PhD², Yon Rojanasakul, PhD^3
1Department of Pharmaceutical Sciences, School of Pharmacy, Hampton University, Hampton, Virginia. ²Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, West Virginia. ³Department of Pharmaceutical and Pharmacological Sciences, School of Pharmacy, West Virginia University, Morgantown, WV

Lung cancer is the leading cause of cancer mortality worldwide. Reactive oxygen/nitrogen species (ROS/RNS) are known to be the key players in lung inflammation and cancer as increased ROS/RNS production leads to genetic mutations, predisposing individuals to cancer. Individuals are constantly exposed to increased levels of ROS/RNS generated from various carcinogens. Hexavalent chromium [Cr(VI)] compounds are redox cycling environmental carcinogens strongly associated with the incidence of lung cancer. Generation of reactive species and dysregulation of apoptosis have been implicated in Cr(VI)-induced lung carcinogenesis; however, the underlying mechanisms are largely unknown. In our study, we observed that ROS, specifically superoxide anion (·O2‾), mediated Cr(VI)-induced apoptosis of human lung epithelial cells. Cr(VI)-induced apoptosis mainly through the mitochondrial death pathway via caspase-9 activation, which was negatively regulated by the mitochondrial anti-apoptotic protein Bcl-2. Specifically, ·O2‾ induced apoptosis by downregulating and degrading Bcl-2 protein through the ubiquitin-proteasomal pathway. Consequently, overexpression of Bcl-2 blocked Cr(VI)-induced apoptosis, whereas Cr(VI)-induced nitric oxide (NO) production was involved in the stabilization of Bcl-2. This mechanism involved NO-mediated S-nitrosylation of Bcl-2, which prevented its ubiquitination and subsequent proteasomal degradation. Therefore, NO was found to exert an anti-apoptotic effect in response to Cr(VI) exposure. The mechanism of Cr(VI)-induced apoptosis resistance was verified in an in vitro model that facilitated mechanistic studies of Cr(VI)-induced carcinogenesis. Long-term exposure to Cr(VI) led to the malignant transformation of non-tumorigenic human lung epithelial cells. Cr(VI)-transformed cells exhibited loss of contact inhibition, colony formation, and increased rates of cell invasion, migration and proliferation as compared to passage-matched control cells. Cr(VI)-transformed cells showed decreased apoptosis and ROS production. The mechanism by which these cells evaded apoptosis involved NO-mediated S-nitrosylation and stabilization of Bcl-2 protein. Taken together, this study establishes an important in vitro model that facilitates mechanistic studies of Cr(VI)-induced carcinogenesis, and elucidates a novel mechanism of apoptosis-resistant and malignant transformation of non-tumorigenic lung cells in response to a human carcinogen. The study also reveals a novel mechanism linking ·O₂‾ and NO with Bcl-2 stability providing a new dimension to reactive species-mediated Bcl-2 stability, apoptotic cell death and cancer development.

Role of Reactive Oxidant Species in Environmental Lung Disease

William N. Rom MD, MPH, Division of Pulmonary and Critical Care Medicine, NYU School of Medicine, New York, NY

ROS contribute to inflammation and injury of the lower respiratory tract after inhalation of particulates that lead to fibrosis and/or cancer. The technique of bronchoalveolar lavage in humans has recovered alveolar macrophages (AMs) in dust diseases that release increased amounts of ROS. Peptide growth factors (PDGF, IGF-I) from AMs and matrix metalloproteinases contribute to the inflammation. Other cells are important: eosinophils cause acute eosinophilic pneumonia due to WTC dust; eosinophils from TPE release very high ROS leading to fibrosis, and neutrophils releasing elastase cause focal emphysema in CWP. Key to the fibrosing process is Transforming Growth Factor-β from myofibroblasts. Myofibroblasts develop from progenitor cells in the basal layers of the airway epithelium and migrate into the interstitium releasing excess amounts of collagen. Progenitor cells activate embryonic signaling pathways through Hedgehog that can be tagged with Gli1-lacZ to identify key players in the ROS-inflammation-fibrosis process.

Cell Signaling and Inflammatory Pathways in Asbestos-Related Diseases

Arti Shukla, PhD, Jedd M. Hillegass, PhD, Maximilian B. MacPherson, BSc, Stacie L. Beuschel, BSc, Sherrill A. Lathrop, BSc, Nicholas H. Heintz, PhD, Brooke T. Mossman, PhD, Department of Pathology, University of Vermont College of Medicine, Burlington, VT

Exposure to asbestos fibers is associated with the development of pulmonary fibrosis, lung cancers and mesotheliomas. Several years ago we questioned the conclusion that asbestos was primarily a DNA- damaging or genotoxic carcinogen, suggesting instead that the initial interaction of asbestos fibers at the plasma membrane triggered multiple signaling cascades that are linked to transcription factors (AP-1, NF-κB, CREB, etc.) governing abnormal cell proliferation and inflammation. Many of these signaling pathways are initiated by elaboration of oxidants both extracellularly and intracellularly. Recently we have shown that asbestos fibers stimulate NADPH oxidases in human macrophages, a key cell in early responses to asbestos, that leads to activation of the inflammasome (Dostert et al, Science 320:674-677). We addressed the hypothesis that NADPH oxidases were over-expressed after exposures to asbestos and in human mesothelioma cells, leading to activation of extracellular-signal regulated kinases (ERKs 1 and 2) critical to the development of asbestos injury, inflammation and mesotheliomas. In RNA interference experiments, human mesothelioma cells (kindly provided by Dr. Harvey I. Pass, NYU) were stably transfected with either shERK1 or shERK2 before characterization in vitro and injection into a mouse xenograft model. We show that ERK2 is causally linked to both asbestos toxicity and the development of mesotheliomas. Microarray experiments on shERK1, shERK2 and shControl mesothelioma lines reveal unique cell death and oxidant/antioxidant genes regulated by ERKs. These studies link ERK mechanistically to the development of inflammation and mesothelioma. (This work is supported by P01 grants from NCI and NHLBI and a training grant from the NIEHS.)

The Role of iNOS-Mediated DNA Damage in Inflammation-Related Carcinogenesis

Shosuke Kawanishi, PhD¹, Yusuke Hiraku, MD, PhD², Takamichi Ichinose, PhD³, Mariko Murata, MD, PhD^2
1Suzuka University of Medical Science, Suzuka, Mie, Japan, ²Mie University Graduate School of Medicine, Mie, Japan, ³Oita University of Nursing and Health Sciences, Oita, Japan

Chronic infection and inflammation considerably contribute to environmental carcinogenesis. Chronic inflammation has been estimated to account for approximately 25 % of human cancers. The International Agency for Research on Cancer (IARC) has estimated that approximately 18 % of cancer cases worldwide is attributable to infectious diseases. Moreover, not only infectious diseases but also various physical, chemical and immunological factors participate in inflammation-related carcinogenesis. Under inflammatory condition, reactive oxygen and nitrogen species are generated from inflammatory and epithelial cells and may play an important role in inflammation-related carcinogenesis by causing DNA damage. Nitric oxide (NO) is generated through the expression of inducible nitric oxide synthase (iNOS) and interacts with superoxide (O2^•-) to form highly reactive peroxynitrite (ONOO-), which reacts with guanine to produce 8-nitroguanine, a mutagenic DNA lesion.

We performed immunohistochemical analysis to examine 8-nitroguanine formation in various clinical specimens and animal models of cancer-prone infectious diseases induced by the liver fluke Opisthorchis viverrini, Helicobacter pylori, hepatitis C virus, human papillomavirus (HPV) and Epstein-Barr virus (EBV). We demonstrated that 8-nitroguanine formation was observed at the sites of carcinogenesis and increased during tumor development. 8-Nitroguanine formation in cervical biopsy specimens of HPV-induced cervical intraepithelial neoplasia was increased with its grade. 8-Nitroguanine formation in cancer cells of patients with EBV-mediated nasopharyngeal carcinoma was significantly stronger than that in biopsy specimens of nasopharyngitis patients. We have also reported that strong 8-nitroguanine formation was closely associated with a poor prognosis in patients with soft tissue tumor.

Asbestos fibers are potent carcinogens causing lung cancer and malignant mesothelioma in humans. We found that 8-nitroguanine was formed particularly in the nucleus of bronchial epithelial cells in the lung tissues of mice intratracheally administered asbestos fibers. Interestingly, quantitative image analysis revealed that crocidolite induced 8-nitrogunaine formation to a significantly greater extent than chrysotile in consistency with their carcinogenic potentials. iNOS expression was also observed in the bronchial epithelial cells and its immunoreactivity was correlated with that of 8-nitrogunaine. On the basis of these findings, we have proposed that 8-nitroguanine could be a potential biomarker to evaluate the risk of inflammation-related carcinogenesis and the prognosis of cancer patients.

Regulation of Apoptosis through Cysteine Oxidation: Implications for Fibrotic Lung Disease

Yvonne MW Janssen-Heininger, PhD¹, Vikas Anathy, PhD¹, Scott Aesif, PhD¹, Jos van der Velden, PhD¹, Amy S. Guala¹, Jessica N. Reiss¹, Elle Roberson¹, Ralph C. Budd², Niki L. Reynaert, PhD³
Department of Pathology¹ and Medicine² University of Vermont, Burlington VT, and Department of Respiratory Medicine³ Maastricht University Medical Center, Maastricht, the Netherlands

Tissue fibrosis is believed to be a manifestation of dysregulated repair following injury, in association with impaired re-epithelialization, and aberrant myofibroblast activation and proliferation. Numerous pathways have been linked to the pathogenesis of fibrotic lung disease, including the death receptor, Fas which contributes to apoptosis of lung epithelial cells. A redox imbalance also has been implicated in disease pathogenesis, although mechanistic details whereby oxidative changes intersect with pro-fibrotic signaling pathways remain elusive. Oxidation of cysteines in proteins, which encompass S-nitrosylation, S-glutathionylation (PSSG), among others are known to act as regulatory events that affect protein structure and function. The overall goal of the research that will be discussed was to determine whether cysteine oxidation events, notably protein S-glutathionylation (PSSG) are important in FasL-induced apoptosis. We recently demonstrated that Fas Ligand (FasL)-induced death of lung epithelial cells is accelerated following S-glutathionylation of the death receptor Fas (Fas-SSG) itself at cysteine 294. Fas-SSG occurs following caspase-8 induced degradation of glutaredoxin, 1, which under physiological conditions acts to decompose PSSG. Fas-SSG promotes its recruitment into lipid rafts, oligomerization, and Death Inducing Signaling Complex formation, leading to feed forward amplification of caspase 8 activation and apoptosis. Ablation of glutaredoxin 1, via SiRNA or gene ablation augments FasL-induced Fas-SSG and sensitizes epithelial cells to apoptosis, while conversely overexpression of glutaredoxin-1 attenuates Fas-SSG, and protects against apoptosis. Using glutaredoxin-1 mediated cysteine derivatization in situ, I will demonstrate evidence that protein S-glutathionylation is increased in two independent mouse models of fibrosis, and in patients with idiopathic pulmonary fibrosis. Lastly, the impact of modulation of glutaredoxin-1 in mouse models will be presented in order to demonstrate the functional significance of protein-S-glutathionylation in fibrogenesis. Funded by NIH R01 HL079331 and R03 HL095404.

Inhibition of Inflammation and Carcinogenesis in the Lung and Colon by Tocopherols

Chung S. Yang, PhD, Guang Xun Li, PhD, Gang Lu, PhD, and Jihyeung Ju, PhD, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ

Tocopherols, which exist in α, β, γ, and δ forms, are antioxidative nutrients known as vitamin E. Although α-tocopherol (α-T) is the major form of vitamin E found in the blood and tissues, γ- and δ-T have been suggested to have stronger anti-inflammatory activities. A robust cancer preventive activity of these tocopherols has not been demonstrated previously. In the present study, using a tocopherol mixture that is rich in γ-T (γ-TmT, which contains 57% γ-T), we demonstrated the inhibition of inflammation as well as of cancer formation and growth in the lung and colon in animal models. When given in the diet at 0.3%, γ-TmT inhibited lung tumorigenesis in the A/J mice, induced by the tobacco carcinogen NNK or NNK plus benzo[a]pyrene (a ubiquitous environmental carcinogen). Dietary 0.3% γ-TmT also inhibited the growth of human lung cancer H1299 cells in xenograft tumors in NCr-nu/nu mice. In both experimental systems, γ-TmT decreased the levels of 8-hydroxydeoxyguanosine (a product of DNA oxidation), γ-H2AX (an indicator of DNA repair caused by DNA double-strand breakage), and nitrotyrosine in tumors. The plasma levels of prostaglandin E2 and leukotriene B4 were also decreased by γ-TmT treatment in mice. More evident anti-inflammatory activity was demonstrated in mice treated with azoxymethane (AOM) and dextran sulfate sodium (DSS), as indicated by the inflammatory index and biochemical markers. The anti-inflammatory activity of pure γ-T and δ-T was also demonstrated in this model. Inhibition of colon carcinogenesis (reduction of adenocarcinoma and adenoma formation by ~80%) was observed when 0.3% γ-TmT diet was initiated either before or after AOM/DSS treatment. These results demonstrate the anti-oxidative, anti-inflammatory, and anti-carcinogenic activities of a mixture of γ-T-rich tocopherols (supported by NIH grant CA122474 and CA120915).

SESSION II: Reactive Oxygen and Reactive Nitrogen Species: Signaling and Detection

Signaling by Reactive Oxygen Species and Aldehydes

Henry Jay Forman, PhD, School of Natural Sciences, University of California, Merced, Merced, CA

Signaling by H₂O₂, α,β-unsaturated aldehydes, such as 4-hydroxy-2-nonenal (HNE) and related chemical species is thought to differ from signaling by other second messengers because the oxidants and other electrophiles can readily undergo non-enzymatic reactions and are therefore classified as “reactive.” Although there are some significant differences in the chemistry of these reactive species from classic second messengers, such as cyclic AMP, there are significant similarities between signaling by H₂O₂ and classic second messengers in terms of the kinetics and regulation of their production and degradation, and effect on signaling pathways including reversibility of action. HNE can form reversible adducts with proteins but its involvement in signaling appears to be less similar to that of the classic second messengers and more like other post-translational signaling, such as farnesylation or ubiquitination.The chemistry of cysteine provides a common factor that underlies signaling by H₂O₂ and HNE. Nonetheless, as H₂O₂ and HNE are rapidly metabolized in vivo and have very different physical properties, spatial considerations are also important in their actions. Therefore, the locations of sources of H₂O₂ and α,β-unsaturated aldehydes, the NADPH oxidases, mitochondria, membrane lipids, and redox cycling toxicants, as well as their targets are key factors. The activation of the JNK pathway by HNE and endogenously generated H₂O₂ illustrates these principles.

S-nitrosylation and Denitrosylation of Proteins in Health and Disease

Jonathan S. Stamler, MD, Duke University, Durham, NC

S-nitrosylation and denitrosylation, the covalent attachment and removal of NO groups from the thiol side chain of cysteine, have emerged as important mechanisms for dynamic, posttranslational regulation of most or all classes of protein. S-nitrosylation thereby conveys a large part of the ubiquitous influence of NO on cellular signal transduction, and provides a prototypic example of redox-based physiological regulation. Accumulating evidence suggests that alterations in S-nitrosylation-regulated signaling contribute to human disease.

Nitric Oxide - A Molecular "Switch" in Redox Signaling

Bruce A. Freeman, PhD, Department of Pharmacology and Chemical Biology, University of Pittsburgh, PA

Over the last 50 years, the posttranslational modification (PTM) of proteins has emerged as a central mechanism for cells to regulate metabolism, growth, differentiation, cell-cell interactions, and immune responses. By influencing protein structure and function, PTM leads to a multiplication of proteome diversity. Redox-dependent PTMs, mediated by environmental and endogenously-generated reactive species, exert both cell signaling and toxicological actions in organisms. PTMs induced by the electrophilic byproducts of redox reactions most frequently occur at protein thiols, with other nucleophilic amino acids serving as less favorable targets. Advances in mass spectrometry and affinity chemistry strategies have improved the detection of electrophile-induced protein modifications both in vitro and in vivo, revealing a high degree of amino acid and protein selectivity of electrophilic PTM. Some PTMs that are induced by electrophilic products of redox reactions appear to modulate physiological signaling pathways that have evolved to act as sensors of oxidative conditions. The identification of these biological targets of electrophiles has motivated further study of the functional impact of various PTM reactions on specific signaling pathways and how this might impact organisms. We also now appreciate that nitric oxide profoundly stimulates the post-translational modification of proteins via the formation of multiple reactive byproducts. This presentation will describe the formation, signaling actions and potential therapeutic uses of electrophilic fatty acid derivatives stemming from these reactions.

NO-Modification of Biomolecules in the Lung Lining: Inflammatory Implications

Andrew Gow, PhD, Rutgers University, Piscataway, NJ

The biological chemistry of Nitric Oxide (NO) is complex in that there are multiple reactive targets and its reactivity is flux rate dependent. However, this very complexity undoubtedly lies at the heart of how NO is capable of controlling such a wide range of biological processes. Nowhere is this more apparent than in the lung where NO controls a wide range of functions ranging from bronchial and vascular tone, to innate immunity and development; and there are multiple reactive targets. These reactive targets allow for the generation of novel NO-modified biomolecules such as S-nitrosylated Surfactant Protein D and nitrated lipids. We have investigated the capability of NO-modified biomolecules to activate cellular signaling pathways in both epithelial and monocytic cells. Our results indicate that these compounds are capable of eliciting a variety of responses and thus may provide a mechanism for the multifarious nature of NO. Specifically, we have begun to investigate how such NO-modifications are involved in a NOS-mediated lung injury model, intratracheal bleomycin administration. Our observations within this model indicate demonstrate the importance of NO-modified biomolecules in mediating lung injury and repair.

Pathophysiological Functions of Reactive Nitrogen Species in Vascular Disorders

Harry Ischiropoulos, PhD, Departments of Pediatrics and Pharmacology, The Children's Hospital of Philadelphia Research Institute and The University of Pennsylvania Philadelphia, PA

Over the last decade basic and translational medicine discoveries have implicated alterations in nitric oxide (NO) metabolism in atherothrombotic diseases. Specifically, we have employed mass-spectrometry-based proteomic approaches to identify site specific NO-mediated oxidative modifications to protein targets of pathobiological relevance to atherothrombotic vascular diseases. Published and new data will be presented relating to the functional consequences of fibrinogen modifications by NO-derived oxidants in animal models of atherosclerosis and in humans. Protein tyrosine nitration also results in the induction of immune responses in humans and animal models. Recent studies have indicated the presence of circulating immunoglobulins that specifically recognize the modified nitrated tyrosine residues in proteins. Preliminary data have indicated that the levels of these specific circulating immunoglobulins are strong predictors of angiographic evidence of coronary artery disease, as well as its acute adverse complications including incident risks for experiencing myocardial infarction, stroke or death. The data suggest that nitrated fibrinogen and immunoglobulins to nitrated proteins may represent two novel prognostic parameters for identifying individuals at increased risk for subsequent adverse cardiac outcomes.

Day 3: Friday, October 30

SESSION V: Role of Oxidants in Nanotoxicology: Screening Tests to Evaluate the Safety of Nanoparticles Prior to Medical Application

Nanotoxicology as a Predictive Science: Use of a Hierarchical Oxidative Stress Screening Paradigm

Andre Nel, MB, ChB, MD, Chief Division of NanoMedicine, Director of UC NanoToxicology Research Training Program and Director of the UC Center for the Environmental Impact of Nanotechnology, University of California, Los Angeles, CA

Because of the large number of new nanomaterials that are being produced, it is of increasing importance to develop a platform for safety and risk assessment. It is not advisable to use the approach for the toxicological assessment of industrial chemicals where only a few hundred of more than 50,000 chemicals has been achieved due to high cost and other logistical constraints. One of the principal stumbling blocks in assessing chemical toxicity has been the cost and time required performing animal and in vivo studies. As a result, new examples of chemical toxicity show up every year, often with devastating consequences to humans and the environment. An intuitively more enlightened approach for nanotechnology would be to develop high throughput screening methods that incorporate a relevant toxicological injury mechanisms that can be related to the physicochemical properties of nanomaterials. I will discuss the emerging paradigms of toxicity that can be linked to the physicochemical properties of engineered nanoparticles with a view to outlining scientific principles that originate at the nano/bio interface and could determine whether interactions fail to occur, are biocompatible or injurious in nature. The major toxicological paradigm that have emerged from nanoparticle toxicity relates to the semiconductor, electronic, UV activation, and redox cycling chemistry of the particles, which allows them to induce tissue damage through the generation of oxygen radicals, electron-hole pairs and oxidant injury. It is possible to follow the oxygen radical generation and oxidant stress injury by abiotic methods as well as a set of hierarchical cellular responses that reflect protective, pro-inflammatory, mitochondrial damaging and pro-apoptotic outcomes. An oxidant injury pathway could translate into adaptive, pro-inflammatory or pro-apoptotic cellular effects in the lung, cardiovascular system, skin and the brain. Another important paradigm relates to the ability of nanoparticles to absorb circulatory or cellular proteins as a function of particle size, surface area, functionalized surface groups, charge, hydrophobicity/hydrophilicity etc. This could induce protein unfolding, protein fibrillation, thiol crosslinking and loss of function, which could lead to neurotoxicity, loss of enzymatic activity, and generation of immunological responses. The thermodynamic properties and free surface energy of nanoparticles as a function of particle size, composition, phase and crystallinity could be responsible for particle dissolution in a biological environment, leading to the generation of cytotoxicity through the release of toxic ions or chemicals. ZnO will be discussed as an example of the latter category. I will demonstrate that it is possible to devise high throughput screening methods to capture these toxicological mechanisms, which can then be used to classify nanoparticles into potentially hazardous and potentially safe. If used as a preliminary screen for newly emerging nanomaterials, these predictive science-based approaches can help to determine which materials should undergo priority testing in animal and in vivo exposure models.

Use of Oxidative Lipidomicsand Signaling by Oxidized Lipids in Safety Screening of Nanoparticles

Valerian E. Kagan, PhD, University of Pittsburgh, Pittsburgh, PA

Fast propagation of nanotechnologies into different industries and consumer products is causing exponential growth of the production of nanomaterials. As a consequence, large amounts of nanomaterials may reach the natural as well as occupational environments thus representing a potential health hazard. Until recently, however, no direct indications of the toxicity of nanoparticles to humans have been reported. A calamitous reminder of possible health hazards associated with the excessive exposure to nanoparticles has been published by Chinese investigators in September, this year of 2009[Song et al.,EurRespir J. 2009 34(3):559-67 ]. They reported sever cases of potential human exposure to polyacrylatenanoparticles - seven young female workers exposed to nanoparticles for 5-13 months; the workers were admitted to a hospital in China with shortness of breath and pleural effusions. Pathological examinations of the patients’ lung tissue revealed nonspecific pulmonary inflammation, pulmonary fibrosis and foreign-body granulomas of pleura. These clinical observations were reminiscent of experimental findings of morphological, biochemical and immunological changes detected after in vivo inhalation exposure of animals to carbonateousnanoparticles [Shvedova et al., 2009]. Exposure of animals to Single-Walled Carbon Nanotubes (SWCNT) is associated with the induction of robust oxidative stress as a component of inflammatory response. Strong oxidants and oxidation reactions are also known to modify SWCNT and cause their degradation. We wondered whether specific enzymatic mechanisms of oxidative stress might be involved in SWCNT biodegradation in physiologically relevant environments. These clinical observations are reminiscent of experimental findings of morphological, biochemical and immunological changes detected after in vivo inhalation exposure of animals to carbonateousnanoparticles [Shvedova et al., 2009].We will present data demonstrating a novel route of biodegradation of SWCNT through enzymatic catalysis by human neutrophil-derived myeloperoxidase (hMPO). Biodegradation was achieved in vitro as well as in human neutrophils; functionalization with immunoglobulin (IgG) promoted cellular internalization of SWCNT and enhanced the hMPO-dependent biodegradation. Molecular modeling suggested an interaction between oxidized sites on carbon nanotubes and residues in proximity to the active site of hMPO. Using an established mouse model of pharyngeal SWCNT aspiration, we showed that biodegradation of carbon nanotubes nullified their ability to induce characteristic pulmonary inflammatory responses. Our findings strongly indicate that numerous novel biomedical applications of carbon nanotubes may be achievable under conditions of carefully controlled biodegradation.

Engineered nanomaterials are becoming a pervasive presence in different spheres of modern life – from diverse technological applications to a plethora of consumer products – and this raises concerns about their possible adverse effects on human health and the environment.¹Among the different nanomaterials available today, carbon nanotubes (CNT), particularly single-walled carbon nanotubes (SWCNT), are most advanced and abundant in their applications. The unique physico-chemical characteristics combined with the vast surface area make the biological effects of CNT and their interactions with cells largely unpredictable.² Therefore, the cytotoxicity of these novel nanomaterials cannot be readily deduced from previous investigations of other particles or fibers. In fact, in vitro data indicate that SWCNT may exert strong cytotoxicity with induction of oxidative stress.^{3, 4, 5} More importantly, recent in vivo studies have demonstrated robust and unusual pulmonary inflammatory responses of SWCNT upon exposure of mice via aspiration or inhalation.^{6, 7, 8} Inflammatory tissue responses were also observed when certain multi-walled CNT with high aspect ratios were administered to mice via intraperitoneal injection.⁹

Carbon Nanotubes

Ken Donaldson, DSc, Craig Poland BSc, Fiona Murphy BSc and Rodger Duffin PhD,
University of Edinburgh, Centre for Inflammation Research, Edinburgh, Scotland

The structure/activity paradigm that explains the pathogenicity of asbestos and other fibres defined the long rigid needle–like shape of the fibres and their ability to persist in the lungs, as requirements for pathogenicity. The essential features of the fibre toxicology paradigm are that to be pathogenic an appreciable number of thin fibres longer than about 15-20µm must enter the lungs and not dissolve/break into shorter fibres. Moving on from our original studies with the peritoneal mesothelium we developed a technique of exposing the pleural mesothelium. We instilled the following samples inrta-pleurally:- two long rigid samples of CNT that looked like asbestos fibres and two CNT samples that were tangled and therefore essentially ‘particles’, rather than fibres; we also used a long-fibre asbestos preparation and a short fibre preparation made from it by milling and a nanoparticle carbon black sample, as controls. Similarly to the peritoneal response, the only samples which caused any evidence of pathogenic effects were those that had long fibres. Tangled and short forms of CNT presented very low or no hazard to the mesothelium whereas long rigid forms of CNT did. The inflammation associated with long nanotubes was persistent at the same level after 7 days as on day one. We developed a hypothesis based on the size-limited clearance of particles through the parietal pleural stomata, that pathogenicity was linked to retention of long fibres in the pleural space. Work on the durability of carbon nanotubes show them to be a highly durable and therefore very likely a highly biopersistent material. Long straight nanotubes therefore fulfil the criteria of the fibre pathogenicity paradigm for being pathogenic i.e. long, thin and biopersistent. We have extended this work to ask questions as to whether other HARN show similar length-dependent toxicity and have confirmed this for Nickel oxide nanowires. We have also been developing in vitro approaches to testing the HARN hazard and an update on this will be supplied.

Acknowledgement. This research was funded by the Colt Foundation and the UK Department of Health

Nanoparticles and the CNS: Dosimetry, Biokinetics, and Effects

Günter Oberdörster, DVM, PhD, University of Rochester, Rochester, NY

Engineered nanoparticles (NPs) are in the same size category as atmospheric ultrafine particles (UFPs), <100 nm. Per given volume, both have high numbers and surface areas compared to larger particles. Epidemiological studies have shown associations between exposure to UFPs and adverse CNS vascular and some cognitive function effects. Exposure to UFP and NPs of experimental animals induced neuronal and inflammatory CNS effects. Access of inhaled NPs to the CNS can occur via sensory nerves existing in the nasopharyngeal and tracheobronchial regions of the respiratory tract and – after translocation into the blood - potentially via the blood brain barrier (BBB). The neuronal pathway circumvents the very tight BBB, although more leaky sections exist in some brain regions. This creates potentially 2 portals of entry into the CNS, involving complex NP-cell-tissue interactions. In general, translocation rates of NP from the portal of entry into the blood compartment or the CNS are very low. Important modifiers of translocation are the physicochemical characteristics of NPs, most notably their size and surface properties, particularly surface chemistry. Primary surface coating (when NPs are manufactured) and secondary surface coating (adsorption of lipids/proteins occurring at the portal of entry and during subsequent translocation) can significantly alter NP biokinetics and their effects. Implications of species differences in respiratory tract anatomy, breathing pattern and brain anatomy for extrapolation to humans of NP effects observed in rodents need to be considered. A causal relationship between long-term exposures to NPs in ambient air (e.g., traffic related) or at the workplace (e.g., metal fumes) and resultant neurotoxic effects in humans has been hypothesized, yet more definitive studies – in particular long term - are needed to test this hypothesis. Some, but probably not the majority of NPs, will have a significant toxicity (hazard) potential, and this will pose a significant risk if there is sufficient exposure of sensitive organs. The challenge is to identify such hazardous NPs and take appropriate measures to prevent exposure.

Pulmonary Toxicity of Single Walled Carbon Nanotubes In Vivo: Relevance to Occupational Exposures

Anna A. Shvedova, PhD, Pathology & Physiology Research Branch/NIOSH/CDC, & Department of Physiology and Pharmacology, WVU, Morgantown, WV

Single-Walled Carbon Nanotubes (SWCNT) are new materials of emerging technological importance and have unique physico-chemical, electronic and mechanical properties. Therefore, they may exhibit unusual interactions with cells and tissues, thus necessitating studies of their toxicity and health effects. We have demonstrated that SWCNT in doses relevant to potential occupational exposures may exert toxic effects in the lung of exposed animals in vivo. We have characterized an unusual and robust inflammatory and fibrogenic response and correlated it with the progression of oxidative stress. We also established that SWCNT induced inflammation and exposure caused altered pulmonary function. Because realistic exposures to SWCNT are likely to occur in conjunction with other pathogenic impacts, e.g., microbial infections, our findings that bacterial clearance from the lungs of SWCNT exposed mice was compromised is of great importance. This talk will address important issues of respiratory toxicity of SWCNT causing substantial pulmonary injury; including fibrosis; results also provide clues elucidation toward underlying mechanisms of toxicity. Acknowledgements: supported by NIOSH OH008282, NORA 92700Y, EC-FP-7-NANOMMUNE-214281. Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health.

SESSION VI: Prevention of Oxygen/Nitrogen Radical Disease

Vascular Inflammation and Oxidative Stress in Atherosclerosis and Aging: Ameliorating Effects of alpha-Lipoic Acid

Balz Frei, PhD, Weijian Zhang, PhD, Lixin Li, PhD, Anthony Smith, PhD, Tory Hagen, PhD, Linus Pauling Institute, Oregon State University, Corvallis, OR

Increased oxidative stress and inflammation are contributing factors to atherosclerotic vascular diseases, for which advanced age is a major risk factor. We have shown previously that the dithiol compound, α-lipoic acid (LA), exerts anti-inflammatory effects by inhibiting tumor necrosis factor-α or lipopolysaccharide (LPS)-induced endothelial cell and monocyte activation in vitro and LPS-induced acute inflammatory responses in vivo. We also reported that LA inhibits atherosclerosis in apolipoprotein E (apoE)-deficient and apoE/LDL receptor-deficient mice, two well-established animal models of human atherosclerosis, in conjunction with significantly less body weight gain, lower serum and VLDL triglyceride levels, and reduced aortic macrophage accumulation and gene expression of adhesion molecules and pro-inflammatory cytokines. In the present study, we investigated the effect of aging and LA on systemic and vascular oxidative stress and inflammation by comparing old (22-24 months) with young (3-4 months) male F344 rats supplemented with LA.

Indicators of oxidative stress, viz., NADPH oxidase activity and superoxide radical levels, were significantly increased in aorta of old compared to young rats, while superoxide dismutase (SOD) activity was decreased. In agreement with the increased NADPH oxidase activity, aortic mRNA and protein levels of the principal catalytic subunit of the enzyme in vascular endothelial cells, NOX4, were markedly increased with age; in contrast, no changes were observed in NOX2 (gp91^phox), the principal catalytic subunit in phagocytic cells, and the other NADPH oxidase catalytic subunit, p22^phox. Aortic enzyme activities of xanthine oxidase and catalase were not different between old and young animals. However, indicators of inflammation, viz., aortic NFκB DNA binding activity and message and protein levels of vascular cell adhesion molecule-1 (VCAM-1), and plasma levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased with age. Dietary supplementation with 0.2% (wt/wt) LA for two weeks caused a non-significant decrease in aortic NADPH oxidase activity in old rats, accompanied by a significant decrease in mRNA–but not protein–levels of NOX4 and VCAM-1. Furthermore, LA supplementation reversed the age-dependent decrease in aortic SOD activity as well as the age-dependent increase in plasma MCP-1 levels.

These data suggest that vascular oxidative stress and inflammation increase with age and LA ameliorates some of these changes, thereby reducing the age-dependent increase in atherosclerotic vascular disease.

This work was supported by grant 0760018Z from the American Heart Association and NIH Center of Excellence grant P01 AT002034.

Oxidant Stress and Airway Disease

David Peden, PhD, The University of North Carolina at Chapel Hill

Oxidant stress is a significant factor in exacerbation of airway disease. Exogenous oxidants like ozone, tobacco smoke, and other particulates are commonly encountered environmental pollutants and are associated with exacerbations of airway disease, like asthma. Biological and nutritional deficits in antioxidant defense may increase risk for pollutant induced airway disease. Null variations in the GSTM1 gene have been associated with increased episodes of asthma exacerbation in heavily polluted areas and in conjunction with tobacco smoke and diesel exhaust challenge. We recently examined the effect of the GSTM1null genotype on response to airway inflammatory responses to ozone in human volunteers. We found that GSTM1 null individuals had increased airway inflammation 24 hours after challenge, including increases in dendritic cell influx. Similar assessments are being conducted in persons following endotoxin challenge. We found that gamma tocopherol inhibits allergen and ozone induced airway inflammation in rodent models. We have also conducted a dosing study of of gamma tocopherol (a variant of vitamin E) in volunteers, and found that in vitro monocyte cytokine response to LPS using peripheral blood mononuclear cells recovered after dosing. Taken together, these studies suggest that genetically defined defects in antioxidant defense may increase risk of oxidant induced lung disease and that antioxidants may play a role in mitigating this effect.

Genes Related to Oxidative Stress and Susceptibility to Air Pollution

Joel Schwartz, PhD, Harvard University, Boston, MA

Airborne particles and ozone are associated with increased morbidity and mortality from heart and lung disease. A host of intermediary biomarkers of effect have also been identified. The mechanistic pathways between exposure and these biomarkers or health events are still being clarified. One hypothesized pathway is oxidative stress, and animal and cell culture data at high exposures clearly implicates this pathway. Epidemiology, in the species of interest and the range of exposure of interest is needed to confirm these findings. Studies of biomarkers of oxidative stress in human populations have begun to do so. An alternative method is to test for functional polymorphisms along the oxidative stress defense pathway, and see if they modify the association between exposure and outcome. These approaches have also been fruitful. Effect modification has been demonstrated for both ozone and particles, and for a range of different outcomes.

Changes in Indicators of Pulmonary Oxidative and Nitrosative Stress Following Drastic Changes in Air Pollution during the Beijing HEART Study

Howard Kipen, Wei Huang, Guangfa Wang, David Rich, Ping Zhu, Yuedan Wang, Min Hu, Shou-En Lu, Pamela Ohman-Strickland, Jicheng Gong, Jian Tong, Tong Zhu, Junfeng Zhang
University of Medicine and Dentistry of New Jersey, Piscataway, NJ; Peking University, Beijing, China

Numerous epidemiologic studies document that daily or hourly changes in air pollution levels, particularly fine particulate matter (PM2.5), are associated with cardiopulmonary morbidity and mortality. However, the precise mechanisms that underlie these associations are not well understood, and further exploration of pollution effects on hypothesized pathogenic pathways, including oxidative stress, is indicated. We present a panel study that examines changes in multiple biomarkers in response to the drastic reductions in Beijing air pollution during the 2008 Olympics (Health Effect of Air Pollution Reduction Trial, HEART). We describe changes in markers of inflammation/nitrosative stress in the lung among 130 healthy Chinese medical students, measured before the Olympics during periods of high pollution and during the Olympics during periods of low pollution. Exhaled nitric oxide (eNO), as well as exhaled breath condensate (EBC), were collected in the morning prior to pollution reductions and then again approximately one month later during the Olympics when reductions in PM2.5 and NO2 ranged from 40-50% below the pre-Olympic levels (~100 µg/m³ and ~ 50 µg/m³, respectively). EBC samples were collected with an ECoScreen II Collector from each subject and then stored until analysis using an HPLC-UV technique. eNO samples were collected offline and analyzed with a chemiluminescence technique. eNO and EBC pH, nitrite and nitrate all showed strong relationships with various indicators of ambient pollution, suggesting a relationship between pollution and the induction of pulmonary oxidative/nitrosative stress in these healthy individuals.

The Future of Antioxidants and the Future of Biomarkers

Barry Halliwell, DSc, National University of Singapore, Singapore

Reactive oxygen species have both beneficial and deleterious effects and are intimately involved in the major human diseases. Yet intervention trials with antioxidant supplements have had, overall, unimpressive effects on disease development and disease progression in human populations. Partly this is because of the lack of effect of “antioxidants” on oxidative damage levels, as measured by “biomarkers” of oxidative stress. Indeed, sometimes “pro-oxidants” may be more effective. One of the most widely used biomarkers is F₂-isoprostanes measurement. Biomarkers must be applied with caution; recent studies on F₂-isoprostane levels in intervention studies and in human disease will be presented to illustrate this.

References
1. Halliwell B and Gutteridge JMC (2007) Free Radicals in Biology and Medicine. Clarendon Press, Oxford (fourth edition), UK.

2. Halliwell B (2009) The wanderings of a free radical. Free Radic Biol Med. 46, 531-542.

3. Halliwell B (2003) Oxidative stress in cell culture: an under-appreciated problem. FEBS Lett. 540, 3-6.

4. Halliwell B (2008). Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies? Arch. Biochem. Biophys. 476, 107-112.

*Additional abstracts coming soon.