Meiying Wang

Use of Metal Oxide Nanoparticle Band Gap To Develop a Predictive Paradigm for Oxidative Stress and Acute Pulmonary Inflammation

We demonstrate for 24 metal oxide (MOx) nanoparticles that it is possible to use conduction band energy levels to delineate their toxicological potential at cellular and whole animal levels. Among the materials, the overlap of conduction band energy (E(c)) levels with the cellular redox potential (-4.12 to -4.84 eV) was strongly correlated to the ability of Co(3)O(4), Cr(2)O(3), Ni(2)O(3), Mn(2)O(3), and CoO nanoparticles to induce oxygen radicals, oxidative stress, and inflammation. This outcome is premised on permissible electron transfers from the biological redox couples that maintain the cellular redox equilibrium to the conduction band of the semiconductor particles. Both single-parameter cytotoxic as well as multi-parameter oxidative stress assays in cells showed excellent correlation to the generation of acute neutrophilic inflammation and cytokine responses in the lungs of C57 BL/6 mice. Co(3)O(4), Ni(2)O(3), Mn(2)O(3), and CoO nanoparticles could also oxidize cytochrome c as a representative redox couple involved in redox homeostasis. While CuO and ZnO generated oxidative stress and acute pulmonary inflammation that is not predicted by E(c) levels, the adverse biological effects of these materials could be explained by their solubility, as demonstrated by ICP-MS analysis. These results demonstrate that it is possible to predict the toxicity of a large series of MOx nanoparticles in the lung premised on semiconductor properties and an integrated in vitro/in vivo hazard ranking model premised on oxidative stress. This establishes a robust platform for modeling of MOx structure-activity relationships based on band gap energy levels and particle dissolution. This predictive toxicological paradigm is also of considerable importance for regulatory decision-making about this important class of engineered nanomaterials.

Nrf2 Is a Key Transcription Factor That Regulates Antioxidant Defense in Macrophages and Epithelial Cells: Protecting against the Proinflammatory and Oxidizing Effects of Diesel Exhaust Chemicals

The proinflammatory effects of particulate pollutants, including diesel exhaust particles (DEP), are related to their content of redox cycling chemicals and their ability to generate oxidative stress in the respiratory tract. An antioxidant defense pathway, which involves phase II enzyme expression, protects against the pro-oxidative and proinflammatory effects of DEP. The expression of enzymes, including heme oxygenase-1 (HO-1) and GST, is dependent on the activity of a genetic antioxidant response element in their promoters. In this study we investigated the mechanism by which redox cycling organic chemicals, prepared from DEP, induce phase II enzyme expression as a protective response. We demonstrate that aromatic and polar DEP fractions, which are enriched in polycyclic aromatic hydrocarbons and quinones, respectively, induce the expression of HO-1, GST, and other phase II enzymes in macrophages and epithelial cells. We show that HO-1 expression is mediated through accumulation of the bZIP transcription factor, Nrf2, in the nucleus, and that Nrf2 gene targeting significantly weakens this response. Nrf2 accumulation and subsequent activation of the antioxidant response element is regulated by the proteasomal degradation of Nrf2. This pathway is sensitive to pro-oxidative and electrophilic DEP chemicals and is also activated by ambient ultrafine particles. We propose that Nrf2-mediated phase II enzyme expression protects against the proinflammatory effects of particulate pollutants in the setting of allergic inflammation and asthma.

Use of Proteomics to Demonstrate a Hierarchical Oxidative Stress Response to Diesel Exhaust Particle Chemicals in a Macrophage Cell Line

Epidemiological studies demonstrate an association between short term exposure to ambient particulate matter (PM) and cardiorespiratory morbidity and mortality. Although the biological mechanisms of these adverse effects are unknown, emerging data suggest a key role for oxidative stress. Ambient PM and diesel exhaust particles (DEP) contain redox cycling organic chemicals that induce pro-oxidative and pro-inflammatory effects in the lung. These responses are suppressed by N-acetylcysteine (NAC), which directly complexes to electrophilic DEP chemicals and exert additional antioxidant effects at the cellular level. A proteomics approach was used to study DEP-induced responses in the macrophage cell line, RAW 264.7. We demonstrate that in the dose range 10–100 μg/ml, organic DEP extracts induce a progressive decline in the cellular GSH/GSSG ratio, in parallel with a linear increase in newly expressed proteins on the two-dimensional gel. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electrospray ionization-liquid chromatography/mass spectrometry/mass spectrometry analysis, 32 newly induced/NAC-suppressed proteins were identified. These include antioxidant enzymes (e.g. heme oxygenase-1 and catalase), pro-inflammatory components (e.g. p38MAPK and Rel A), and products of intermediary metabolism that are regulated by oxidative stress. Heme oxygenase-1 was induced at low extract dose and with minimal decline in the GSH/GSSG ratio, whereas MAP kinase activation required a higher chemical dose and incremental levels of oxidative stress. Moreover, at extract doses >50 μg/ml, there is a steep decline in cellular viability. These data suggest that DEP induce a hierarchical oxidative stress response in which some of these proteins may serve as markers for oxidative stress during PM exposures.

Comparison of the Pro-Oxidative and Proinflammatory Effects of Organic Diesel Exhaust Particle Chemicals in Bronchial Epithelial Cells and Macrophages

Inhaled diesel exhaust particles (DEP) exert proinflammatory effects in the respiratory tract. This effect is related to the particle content of redox cycling chemicals and is involved in the adjuvant effects of DEP in atopic sensitization. We demonstrate that organic chemicals extracted from DEP induce oxidative stress in normal and transformed bronchial epithelial cells, leading to the expression of heme oxygenase 1, activation of the c-Jun N-terminal kinase cascade, IL-8 production, as well as induction of cytotoxicity. Among these effects, heme oxygenase 1 expression is the most sensitive marker for oxidative stress, while c-Jun N-terminal kinase activation and induction of apoptosis-necrosis require incremental amounts of the organic chemicals and increased levels of oxidative stress. While a macrophage cell line (THP-1) responded in similar fashion, epithelial cells produced more superoxide radicals and were more susceptible to cytotoxic effects than macrophages. Cytotoxicity is the result of mitochondrial damage, which manifests as ultramicroscopic changes in organelle morphology, a decrease in the mitochondrial membrane potential, superoxide production, and ATP depletion. Epithelial cells also differ from macrophages in not being protected by a thiol antioxidant, N-acetylcysteine, which effectively protects macrophages against cytotoxic DEP chemicals. These findings show that epithelial cells exhibit a hierarchical oxidative stress response that differs from that of macrophages by more rapid transition from cytoprotective to cytotoxic responses. Moreover, epithelial cells are not able to convert N-acetylcysteine to cytoprotective glutathione.

Use of a stratified oxidative stress model to study the biological effects of ambient concentrated and diesel exhaust particulate matter.

Although several epidemiological studies have shown a positive relationship between exposure to ambient air particulate matter (PM) and adverse health effects in humans, there is still a fundamental lack of understanding of the most toxic particle components and the biological mechanisms through which they act. Since our studies on the biological effects of diesel exhaust particles (DEP) have highlighted the role of reactive oxygen species (ROS), catalyzed by organic chemical compounds, we set out to establish whether this constitutes an oxidative stress model that can be used to study the biological effects of ambient coarse and fine PM. We demonstrate that organic DEP extracts induce a stratified oxidative stress response, leading to heme oxygenase 1 (HO-1) expression at normal GSH/ GSSG ratios, proceed to Jun kinase activation and interleukin 8 (IL-8) production at intermediary oxidative stress levels, and culminate in cellular apoptosis in parallel with a sharp decline in GSH/GSSG ratios. We demonstrate that ambient concentrated air particulates, collected with a particle concentrator and a liquid impinger, mimic the effects of organic DEP extracts at lower oxidative stress levels. While fine PM consistently induced HO-1 expression in all most of the samples collected over a 9-mo survey period, coarse particulates were effective at inducing that effect during fall and winter. Moreover, HO-1 expression was positively correlated to the higher organic carbon (OC) and polyaromatic hydrocarbons (PAHs) content of fine versus coarse PM, as well as the rise in PAH content that occurs in coarse PM during the winter months. Although coarse and fine PM lead to a decrease in cellular glutathione (GSH)/GSSG ratios, oxidative stress did not increase to cytotoxic levels. Taken together, these data demonstrate that it is possible to use the stratified oxidative stress model developed for DEP to interpret the biological effects of coarse and fine PM. This work has important implications for the selection of relevant biological endpoints for in vivo studies.

Decreased Dissolution of ZnO by Iron Doping Yields Nanoparticles with Reduced Toxicity in the Rodent Lung and Zebrafish Embryos

We have recently shown that the dissolution of ZnO nanoparticles and Zn(2+) shedding leads to a series of sublethal and lethal toxicological responses at the cellular level that can be alleviated by iron doping. Iron doping changes the particle matrix and slows the rate of particle dissolution. To determine whether iron doping of ZnO also leads to lesser toxic effects in vivo, toxicity studies were performed in rodent and zebrafish models. First, we synthesized a fresh batch of ZnO nanoparticles doped with 1-10 wt % of Fe. These particles were extensively characterized to confirm their doping status, reduced rate of dissolution in an exposure medium, and reduced toxicity in a cellular screen. Subsequent studies compared the effects of undoped to doped particles in the rat lung, mouse lung, and the zebrafish embryo. The zebrafish studies looked at embryo hatching and mortality rates as well as the generation of morphological defects, while the endpoints in the rodent lung included an assessment of inflammatory cell infiltrates, LDH release, and cytokine levels in the bronchoalveolar lavage fluid. Iron doping, similar to the effect of the metal chelator, DTPA, interfered in the inhibitory effects of Zn(2+) on zebrafish hatching. In the oropharyngeal aspiration model in the mouse, iron doping was associated with decreased polymorphonuclear cell counts and IL-6 mRNA production. Doped particles also elicited decreased heme oxygenase 1 expression in the murine lung. In the intratracheal instillation studies in the rat, Fe doping was associated with decreased polymorphonuclear cell counts, LDH, and albumin levels. All considered, the above data show that Fe doping is a possible safe design strategy for preventing ZnO toxicity in animals and the environment.

Thiol Antioxidants Inhibit the Adjuvant Effects of Aerosolized Diesel Exhaust Particles in a Murine Model for Ovalbumin Sensitization

Although several epidemiological studies indicate a correlation between exposure to ambient particulate matter and adverse health effects in humans, there is still a fundamental lack of understanding of the mechanisms involved. We set out to test the hypothesis that reactive oxygen species are involved in the adjuvant effects of diesel exhaust particles (DEP) in a murine OVA sensitization model. First, we tested six different antioxidants, N-acetylcysteine (NAC), bucillamine (BUC), silibinin, luteolin, trolox (vitamin E), and ascorbic acid, for their ability to interfere in DEP-mediated oxidative stress in vitro. Of the six agents tested, only the thiol antioxidants, BUC and NAC, were effective at preventing a decrease in intracellular reduced glutathione:glutathione disulfide ratios, protecting cells from protein and lipid oxidation, and preventing heme oxygenase 1 expression. Therefore, we selected the thiol antioxidants for testing in the murine OVA inhalation sensitization model. Our data demonstrate that NAC and BUC effectively inhibited the adjuvant effects of DEP in the induction of OVA-specific IgE and IgG1 production. Furthermore, NAC and BUC prevented the generation of lipid peroxidation and protein oxidation in the lungs of OVA- plus DEP-exposed animals. These findings indicate that NAC and BUC are capable of preventing the adjuvant effects of inhaled DEP and suggest that oxidative stress is a key mechanistic component in the adjuvant effect of DEP. Antioxidant treatment strategies may therefore serve to alleviate allergic inflammation and may provide a rational basis for treating the contribution of particulate matter to asthmatic disease.

The adjuvant effect of ambient particulate matter is closely reflected by the particulate oxidant potential

Background It has been demonstrated that ambient particulate matter (PM) can act as an adjuvant for allergic sensitization. Redox-active organic chemicals on the particle surface play an important role in PM adverse health effects and may determine the adjuvant effect of different particle types according to their potential to perturb redox equilibrium in the immune system. Objectives We determined whether the adjuvant effect of ambient fine particles versus ultrafine particles (UFPs) is correlated to their prooxidant potential. Methods We have established an intranasal sensitization model that uses ambient PM as a potential adjuvant for sensitization to ovalbumin (OVA), which enhances the capacity for secondary OVA challenge to induce allergic airway inflammation. Results UFPs with a greater polycyclic aromatic hydrocarbon (PAH) content and higher oxidant potential enhanced OVA sensitization more readily than did fine particles. This manifests as enhanced allergic inflammation upon secondary OVA challenge, leading to eosinophilic inflammation and mucoid hyperplasia starting at the nasal turbinates all the way down to the small pulmonary airways. The thiol antioxidant N-acetyl cysteine was able to suppress some of these sensitization events. Conclusions The adjuvant effects of ambient UFP is determined by their oxidant potential, which likely plays a role in changing the redox equilibrium in the mucosal immune system.

Use of Coated Silver Nanoparticles to Understand the Relationship of Particle Dissolution and Bioavailability to Cell and Lung Toxicological Potential

Since more than 30% of consumer products that include engineered nanomaterials contain nano-Ag, the safety of this material is of considerable public concern. In this study, Ag nanoparticles (NPs) are used to demonstrate that 20 nm polyvinylpyrrolidone (PVP or P) and citrate (C)-coated Ag NPs induce more cellular toxicity and oxidative stress than larger (110 nm) particles due to a higher rate of dissolution and Ag bioavailability. Moreover, there is also a higher propensity for citrate 20 nm (C20) nanoparticles to generate acute neutrophilic inflammation in the lung and to produce chemokines compared to C110. P110 has less cytotoxic effects than C110, likely due to the ability of PVP to complex released Ag(+) . In contrast to the more intense acute pulmonary effects of C20, C110 induces mild pulmonary fibrosis at day 21, likely as a result of slow but persistent Ag(+) release leading to a sub-chronic injury response. Interestingly, the released metallic Ag is incorporated into the collagen fibers depositing around airways and the lung interstitium. Taken together, these results demonstrate that size and surface coating affect the cellular toxicity of Ag NPs as well as their acute versus sub-chronic lung injury potential.

Use of a Lipid-Coated Mesoporous Silica Nanoparticle Platform for Synergistic Gemcitabine and Paclitaxel Delivery to Human Pancreatic Cancer in Mice

Recently, a commercial albumin-bound paclitaxel (PTX) nanocarrier (Abraxane) was approved as the first new drug for pancreatic ductal adenocarcinoma in almost a decade. PTX improves the pharmaceutical efficacy of the first-line pancreatic cancer drug, gemcitabine (GEM), through suppression of the tumor stroma and inhibiting the expression of the GEM-inactivating enzyme, cytidine deaminase (CDA). We asked, therefore, whether it was possible to develop a mesoporous silica nanoparticle (MSNP) carrier for pancreatic cancer to co-deliver a synergistic GEM/PTX combination. High drug loading was achieved by a custom-designed coated lipid film technique to encapsulate a calculated dose of GEM (40 wt %) by using a supported lipid bilayer (LB). The uniform coating of the 65 nm nanoparticles by a lipid membrane allowed incorporation of a sublethal amount of hydrophobic PTX, which could be co-delivered with GEM in pancreatic cells and tumors. We demonstrate that ratiometric PTX incorporation and delivery by our LB-MSNP could suppress CDA expression, contemporaneous with induction of oxidative stress as the operating principle for PTX synergy. To demonstrate the in vivo efficacy, mice carrying subcutaneous PANC-1 xenografts received intravenous (IV) injection of PTX/GEM-loaded LB-MSNP. Drug co-delivery provided more effective tumor shrinkage than GEM-loaded LB-MSNP, free GEM, or free GEM plus Abraxane. Comparable tumor shrinkage required coadministration of 12 times the amount of free Abraxane. High-performance liquid chromatography analysis of tumor-associated GEM metabolites confirmed that, compared to free GEM, MSNP co-delivery increased the phosphorylated DNA-interactive GEM metabolite 13-fold and decreased the inactivated and deaminated metabolite 4-fold. IV injection of MSNP-delivered PTX/GEM in a PANC-1 orthotopic model effectively inhibited primary tumor growth and eliminated metastatic foci. The enhanced in vivo efficacy of the dual delivery carrier could be achieved with no evidence of local or systemic toxicity. In summary, we demonstrate the development of an effective LB-MSNP nanocarrier for synergistic PTX/GEM delivery in pancreatic cancer.