Stephen S. Leonard

Shape-enhanced Photocatalytic Activity of Single-crystalline Anatase TiO2 (101) Nanobelts

Particle size is generally considered to be the primary factor in the design of nanocrystal photocatalysts, because the reduction of particle size increases the number of active sites. However, the benefit from the size reduction can be canceled by a higher electron-hole recombination rate due to the confined space in sphere-shaped nanoparticles. Here we report a mechanistic study on a novel nanobelt structure that overcomes the drawback of sphere-shaped nanoparticles. Single-crystalline anatase TiO(2) nanobelts with two dominant surfaces of (101) facet exhibit enhanced photocatalytic activity over the nanosphere counterparts with an identical crystal phase and similar specific surface area. The ab initio density functional theory (DFT) calculations show that the exposed (101) facet of the nanobelts yields an enhanced reactivity with molecular O(2), facilitating the generation of superoxide radical. Moreover, the nanobelts exhibit a lower electron-hole recombination rate than the nanospheres due to the following three reasons: (i) greater charge mobility in the nanobelts, which is enabled along the longitudinal dimension of the crystals; (ii) fewer localized states near the band edges and in the bandgap due to fewer unpassivated surface states in the nanobelts; and (iii) enhanced charge separation due to trapping of photogenerated electrons by chemisorbed molecular O(2) on the (101) facet. Our results suggest that the photocatalysis efficiency of nanocrystals can be significantly improved by tailoring the shape and the surface structure of nanocrystals, which provides a new concept for rational design and development of high-performance photocatalysts.

Mouse pulmonary dose- and time course-responses induced by exposure to multi-walled carbon nanotubes

Carbon nanotubes (CNT) come in a variety of types, but one of the most common forms is multi-walled carbon nanotubes (MWCNT). MWCNT have potential applications in many diverse commercial processes, and thus human exposures are considered to be likely. In order to investigate the pulmonary toxicity of MWCNT, we conducted an in vivo dose-response and time course study of MWCNT in mice in order to assess their ability to induce pulmonary inflammation, damage, and fibrosis using doses that approximate estimated human occupational exposures. MWCNT were dispersed in dispersion medium (DM) and male C57BL/6J mice (7 weeks old) received either DM (vehicle control), 10, 20, 40 or 80mug MWCNT by aspiration exposure. At 1, 7, 28 and 56 days post-exposure, MWCNT-induced pulmonary toxicity was investigated. Bronchoalveolar lavage (BAL) studies determined pulmonary inflammation and damage was dose-dependent and peaked at 7 days post-exposure. By 56 days post-exposure, pulmonary inflammation and damage markers were returning to control levels, except for the 40mug MWCNT dose, which was still significantly higher than vehicle control. Histopathological studies determined that MWCNT exposure caused rapid development of pulmonary fibrosis by 7 days post-exposure, that granulomatous inflammation persisted throughout the 56-day post-exposure period, and also demonstrated that MWCNT can reach the pleura after pulmonary exposure. In summary, the data reported here indicate that MWCNT exposure rapidly produces significant adverse health outcomes in the lung. Furthermore, the observation that MWCNT reach the pleura after aspiration exposure indicates that more extensive investigations are needed to fully assess if pleural penetration results in any adverse health outcomes.

Cardiovascular Effects of Pulmonary Exposure to Single-Wall Carbon Nanotubes

Background Engineered nanosized materials, such as single-wall carbon nanotubes (SWCNT), are emerging as technologically important in different industries. Objective The unique physical characteristics and the pulmonary toxicity of SWCNTs raised concerns that respiratory exposure to these materials may be associated with cardiovascular adverse effects. Methods In these studies we evaluated aortic mitochondrial alterations by oxidative stress assays, including quantitative polymerase chain reaction of mitochondrial (mt) DNA and plaque formation by morphometric analysis in mice exposed to SWCNTs. Results A single intrapharyngeal instillation of SWCNTs induced activation of heme oxygenase-1 (HO-1), a marker of oxidative insults, in lung, aorta, and heart tissue in HO-1 reporter transgenic mice. Furthermore, we found that C57BL/6 mice, exposed to SWCNT (10 and 40 μg/mouse), developed aortic mtDNA damage at 7, 28, and 60 days after exposure. mtDNA damage was accompanied by changes in aortic mitochondrial glutathione and protein carbonyl levels. Because these modifications have been related to cardiovascular diseases, we evaluated whether repeated exposure to SWCNTs (20 μg/mouse once every other week for 8 weeks) stimulates the progression of atherosclerosis in ApoE−/− transgenic mice. Although SWCNT exposure did not modify the lipid profiles of these mice, it resulted in accelerated plaque formation in ApoE−/− mice fed an atherogenic diet. Plaque areas in the aortas, measured by the en face method, and in the brachiocephalic arteries, measured histopathologically, were significantly increased in the SWCNT-treated mice. This response was accompanied by increased mtDNA damage but not inflammation. Conclusions Taken together, the findings are of sufficient significance to warrant further studies to evaluate the systemic effects of SWCNT under workplace or environmental exposure paradigms.

Concept of Assessing Nanoparticle Hazards Considering Nanoparticle Dosemetric and Chemical/Biological Response Metrics

Engineered nanoparticles (NP) are being developed and incorporated in a number of commercial products, raising the potential of human exposure during manufacture, use, and disposal. Although data concerning the potential toxicity of some NP have been reported, validated simple assays are lacking for predicting their in vivo toxicity. The aim of this study was to evaluate new response metrics based on chemical and biological activity of NP for screening assays that can be used to predict NP toxicity in vivo. Two cell-free and two cell-based assays were evaluated for their power in predicting in vivo toxicity of eight distinct particle types with widely differing physicochemical characteristics. The cell-free systems comprised fluorescence- and electron spin resonance-based assays of oxidant activity. The cell-based systems also used electron spin resonance (ESR) as well as luciferase reporter activity to rank the different particle types in comparison to benchmark particles of low and high activity. In vivo experiments evaluated acute pulmonary inflammatory responses in rats. Endpoints in all assays were related to oxidative stress and responses were expressed per unit NP surface area to compare the results of different assays. Results indicated that NP are capable of producing reactive species, which in biological systems lead to oxidative stress. Copper NP had the greatest activity in all assays, while TiO2 and gold NP generally were the least reactive. Differences in the ranking of NP activity among the assays were found when comparisons were based on measured responses. However, expressing the chemical (cell-free) and biological (cells; in vivo) activity per unit particle surface area showed that all in vitro assays correlated significantly with in vivo results, with the cellular assays correlating the best. Data from this study indicate that it is possible to predict acute in vivo inflammatory potential of NP with cell-free and cellular assays by using NP surface area-based dose and response metrics, but that a cellular component is required to achieve a higher degree of predictive power.

Vanadate-induced Expression of Hypoxia-inducible Factor 1α and Vascular Endothelial Growth Factor through Phosphatidylinositol 3-Kinase/Akt Pathway and Reactive Oxygen Species

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1α and HIF-1β/aryl hydrocarbon nuclear translocator subunits. HIF-1 expression is induced by hypoxia, growth factors, and activation of oncogenes. In response to hypoxia, HIF-1 activates the expression of many genes including vascular endothelial growth factor (VEGF) and erythropoietin. HIF-1 and VEGF play an important role in angiogenesis and tumor progression. Vanadate is widely used in industry, and is a potent inducer of tumors in humans and animals. In this study, we demonstrate that vanadate induces HIF-1 activity through the expression of HIF-1α but not HIF-1β subunit, and increases VEGF expression in DU145 human prostate carcinoma cells. We also studied the signaling pathway involved in vanadate-induced HIF-1α and VEGF expression and found that phosphatidylinositol 3-kinase/Akt signaling was required for HIF-1 and VEGF expression induced by vanadate, whereas mitogen-activated protein kinase pathway was not required. We also found that reactive oxygen species (ROS) were involved in vanadate-induced expression of HIF-1 and VEGF in DU145 cells. The major species of ROS responsible for the induction of HIF-1 and VEGF expression was H2O2. These results suggest that the expression of HIF-1 and VEGF induced by vanadate through PI3K/Akt may be an important signaling pathway in the vanadate-induced carcinogenesis, and ROS may play an important role.

S-Nitrosylation of Bcl-2 Inhibits Its Ubiquitin-Proteasomal Degradation A NOVEL ANTIAPOPTOTIC MECHANISM THAT SUPPRESSES APOPTOSIS

Bcl-2 is a key apoptosis regulatory protein of the mitochondrial death pathway whose function is dependent on its expression levels. Although Bcl-2 expression is controlled by various mechanisms, post-translational modifications, such as ubiquitination and proteasomal degradation, have emerged as important regulators of Bcl-2 function. However, the underlying mechanisms of this regulation are unclear. We report here that Bcl-2 undergoes S-nitrosylation by endogenous nitric oxide (NO) in response to multiple apoptotic mediators and that this modification inhibits ubiquitin-proteasomal degradation of Bcl-2. Inhibition of NO production by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and by NO synthase inhibitor aminoguanidine effectively inhibited S-nitrosylation of Bcl-2, increased its ubiquitination, and promoted apoptotic cell death induced by chromium (VI). In contrast, the NO donors dipropylenetriamine NONOate and sodium nitroprusside showed opposite effects. The effect of NO on Bcl-2 stability was shown to be independent of its dephosphorylation. Mutational analysis of Bcl-2 further showed that the two cysteine residues of Bcl-2 (Cys158 and Cys229) are important in the S-nitrosylation process and that mutations of these cysteines completely inhibited Bcl-2 S-nitrosylation. Treatment of the cells with other stress inducers, including Fas ligand and buthionine sulfoxide, also induced Bcl-2 S-nitrosylation, suggesting that this is a general phenomenon that regulates Bcl-2 stability and function under various stress conditions. These findings indicate a novel function of NO and its regulation of Bcl-2, which provides a key mechanism for the control of apoptotic cell death and cancer development.

Vanadate Induces p53 Transactivation through Hydrogen Peroxide and Causes Apoptosis

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms controlling vanadate-induced adverse effects remain to be elucidated. The present study investigated the vanadate-induced p53 activation and involvement of reactive oxygen species (ROS) in p53 activation as well as the role of p53 in apoptosis induction by vanadate. Exposure of mouse epidermal JB6 cells to vanadate led to transactivation of p53 activity in a time- and dose-dependent manner. It also caused mitochondrial damage, apoptosis, and generated ROS. Scavenging of vanadate-induced H2O2 byN-acetyl-l-cysteine (a general antioxidant) or catalase (a specific H2O2 inhibitor), or the chelation of vanadate by deferoxamine, resulted in inhibition of p53 activation and cell mitochondrial damage. In contract, an increase in H2O2 generation in response to superoxide dismutase or NADPH enhanced these effects caused by vanadate. Furthermore, vanadate-induced apoptosis occurred in cells expressing wild-type p53 (p53+/+) but was very weak in p53-deficient (p53−/−) cells. These results demonstrate that vanadate induces p53 activation mainly through H2O2 generation, and this activation is required for vanadate-induced apoptosis.

Acute pulmonary dose–responses to inhaled multi-walled carbon nanotubes

Abstract This study investigated the in vivo pulmonary toxicity of inhaled multi-walled carbon nanotubes (MWCNT). Mice-inhaled aerosolized MWCNT (10 mg/m3, 5 h/day) for 2, 4, 8 or 12 days. MWCNT lung burden was linearly related to exposure duration. MWCNT-induced pulmonary inflammation was assessed by determining whole lung lavage (WLL) polymorphonuclear leukocytes (PMN). Lung cytotoxicity was assessed by WLL fluid LDH activities. WLL fluid albumin concentrations were determined as a marker of alveolar air–blood barrier integrity. These parameters significantly increased in MWCNT-exposed mice versus controls and were dose-dependent. Histopathologic alterations identified in the lung included (1) bronciolocentric inflammation, (2) bronchiolar epithelial hyperplasia and hypertrophy, (3) fibrosis, (4) vascular changes and (5) rare pleural penetration. MWCNT translocated to the lymph node where the deep paracortex was expanded after 8 or 12 days. Acute inhalation of MWCNT induced dose-dependent pulmonary inflammation and damage with rapid development of pulmonary fibrosis, and also demonstrated that MWCNT can reach the pleura after inhalation exposure.

Role of reactive oxygen species and MAPKs in vanadate-induced G2/M phase arrest

Cell growth arrest is an important mechanism in maintaining genomic stability and integrity in response to environmental stress. Using the human lung alveolar epithelial cancer cell line A549, we investigated the role of reactive oxygen species (ROS), extracellular signal-regulated protein kinase (ERK), and p38 protein kinase in vanadate-induced cell growth arrest. Exposure of cells to vanadate led to cell growth arrest at the G(2)/M phase and caused upregulation of p21 and phospho-cdc2 and degradation of cdc25C in a time- and dose-dependent manner. Vanadate stimulated mitogen-activated protein kinases (MAPKs) family members, as determined by the phosphorylation of ERK and p38. PD98059, an inhibitor of ERK, and SB202190, an inhibitor of p38, inhibited vanadate-induced cell growth arrest, upregulation of p21 and cdc2, and degradation of cdc25C. In addition to hydroxyl radical ((*)OH) formation, cellular reduction of vanadate generated superoxide radical (O(2)(*)(-)) and hydrogen peroxide (H(2)O(2)), as determined by confocal microscopy using specific dyes. Generation of O(2)(*)(-) and H(2)O(2) was inhibited by specific antioxidant enzymes, superoxide dismutase (SOD) and catalase, respectively. ROS activate ERK and p38, which in turn upregulate p21 and cdc2 and cause degradation of cdc25C, leading to cell growth arrest at the G(2)/M phase. Specific ROS affect different MAPK family members and cell growth regulatory proteins with different potencies.

Antioxidant properties of aspirin: Characterization of the ability of aspirin to inhibit silica-induced lipid peroxidation, DNA damage, NF-κB activation, and TNF-α production

Electron spin resonance (ESR) was used to investigate the reaction of aspirin toward reactive oxygen species, such as hydroxyl radicals (·OH), superoxide radicals (O2-) and H2O2. The Fenton reaction (Fe(II) + H2O2 ---> FE(III) + -OH + OR) was used as a source of -OH radicals. The results show that aspirin is an efficient -OH radical scavenger with a reaction rate constant of k = 3.6 x 1010 M-1sec-1, which is faster than several well established antioxidants, such as ascorbate, glutathione and cysteine. However, aspirin is not a good scavenger for O2- or H2O2. Through its antioxidant property, aspirin exhibited a protective effect against silica-induced lipid peroxidation and DNA strand breakage. Aspirin also inhibited the activation of nuclear transcription factor-κb induced by silica, lipopolysaccharide or the transition metal, Fe(II), as demonstrated by electrophoretic mobility shift assay. The results show that aspirin functions as an antioxidant via its ability to scavenge -OH radicals. This antioxidant property may explain some of its various physiological and pharmacological actions.