Marine Bile Natural Products as Agonists of the TGR5 Receptor.

Agonism of the G protein-coupled bile acid receptor "Takeda G-protein receptor 5" (TGR5) aids in attenuating cholesterol accumulation due to atherosclerotic progression. Although mammalian bile compounds can activate TGR5, they are generally weak agonists, and more effective compounds need to be identified. In this study, two marine bile compounds (5ß-scymnol and its sulfate) were compared with mammalian bile compounds deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) using an in vitro model of TGR5 agonism. The response profiles of human embryonic kidney 293 cells (HEK293) transfected to overexpress TGR5 (HEK293-TGR5) and incubated with subcytotoxic concentrations of test compounds were compared to nontransfected HEK293 control cells using the specific calcium-binding fluorophore Fura-2AM to measure intracellular calcium [Ca2+]i release. Scymnol and scymnol sulfate caused a sustained increase in [Ca2+]i within TGR5 cells only, which was abolished by a specific inhibitor for Gαq protein (UBO-QIC). Sustained increases in [Ca2+]i were seen in both cell types with DCA exposure; this was unaffected by UBO-QIC, indicating that TGR5 activation was not involved. Exposure to UDCA did not alter [Ca2+]i, suggesting a lack of TGR5 bioactivity. These findings demonstrated that both scymnol and scymnol sulfate are novel agonists of TGR5 receptors, showing therapeutic potential for treating atherosclerosis.

An advanced method for quantitative measurements of cholesterol crystallization.

BACKGROUND: Cholesterol crystallization within an atherosclerotic plaque significantly contributes to the acceleration of plaque rupture - a problematic event due to the current lack of specific treatments to prevent such formations. Modelling this pathogenic process is also difficult due to the lack of suitable experimental models that enable quantitative analysis of crystal formation and bioactivity screening of potential therapeutic compounds. AIM: To develop an in vitro human cell model of cholesterol crystallization combined with an imaging system that incorporates both quantitative analysis and real-time continuous imaging of cholesterol crystal formation. METHODS AND RESULTS: An enhanced in vitro model of cholesterol crystallization was developed through the use of acetylated low-density lipoprotein (AcLDL) and 7-ketocholesterol as agents of foam cell induction within a human THP-1 monocytic cell line. Advanced confocal and polarizing microscopies were incorporated into the model so as to allow for quantitation of cholesterol crystallization, with the lipid-loaded group producing significantly greater numbers of cholesterol crystals than the untreated group. The utility of this system was also demonstrated by investigating the effects of the cholesterol-lowering drug lovastatin and therapeutic bile compound ursodeoxycholic acid (UDCA), showing that these drugs influence different aspects of cholesterol crystal formation. CONCLUSIONS: The in vitro human THP-1 monocyte model of cholesterol crystallization provides an effective and efficient means of quantitating cholesterol crystallization in the pre-clinical stage of research. The model also allows for the screening of potentially therapeutic compounds that may be used in attenuating or preventing cholesterol crystallization.

Samarium doped titanium dioxide nanoparticles as theranostic agents in radiation therapy.

PURPOSE: Titanium dioxide nanoparticles (TiO2 NPs) have been investigated for their role as radiosensitisers for radiation therapy. The study aims to increase the efficiency of these NPs by synthesising them with samarium. METHODS: Samarium-doped TiO2 NPs (Ti(Sm)O2 NPs) were synthesised using a solvothermal method. Transmission electron microscopy (TEM), X-ray diffraction (XRD), and energy-dispersive X-ray spectroscopy (EDS) were performed for characterising of the Ti(Sm)O2 NPs. The intracellular uptake and cytotoxicity were assessed in vitro using A549 and DU145 cancer cell lines. Furthermore, the effect of dose enhancement and generation of reactive oxygen species (ROS) in response to 6 MV X-rays was evaluated. Additionally, the image contrast properties were investigated using computed tomography (CT) images. RESULTS: The synthesised Ti(Sm)O2 NPs were about 13 nm in diameter as determined by TEM. The XRD pattern of Ti(Sm)O2 NPs was consistent with that of anatase-type TiO2. EDS confirmed the presence of samarium in the nanoparticles. At 200 µg/ml concentration, no differences in cellular uptake and cytotoxicity were observed between TiO2 NPs and Ti(Sm)O2 NPs in both A549 and DU145 cells. However, the combination of Ti(Sm)O2 NPs and X-rays elicited higher cytotoxic effect and ROS generation in the cells than that with TiO2 NPs and X-rays. The CT numbers of Ti(Sm)O2 NPs were systematically higher than that of TiO2 NPs. CONCLUSIONS: The Ti(Sm)O2 NPs increased the dose enhancement of MV X-ray beams than that elicited by TiO2 NPs. Samarium improved the efficiency of TiO2 NPs as potential radiosensitising agent.

Combined Effects of Gold Nanoparticles and Ionizing Radiation on Human Prostate and Lung Cancer Cell Migration.

The effect of 15 nm-sized gold nanoparticles (AuNPs) and/or ionizing radiation (IR) on the migration and adhesion of human prostate (DU145) and lung (A549) cancer cell lines was investigated. Cell migration was measured by observing the closing of a gap created by a pipette tip on cell monolayers grown in 6-well plates. The ratio of the gap areas at 0 h and 24 h were used to calculate the relative migration. The relative migration of cells irradiated with 5 Gy was found to be 89% and 86% for DU145 and A549 cells respectively. When the cells were treated with 1 mM AuNPs this fell to ~75% for both cell lines. However, when the cells were treated with both AuNPs and IR an additive effect was seen, as the relative migration rate fell to ~60%. Of interest was that when the cells were exposed to either 2 or 5 Gy IR, their ability to adhere to the surface of a polystyrene culture plate was significantly enhanced, unlike that seen for AuNPs. The delays in gap filling (cell migration) in cells treated with IR and/or AuNPs can be attributed to cellular changes which also may have altered cell motility. In addition, changes in the cytoskeleton of the cancer cells may have also affected adhesiveness and thus the cancer cell's motility response to IR.

ZnO nanoparticles and organic chemical UV-filters are equally well tolerated by human immune cells.

An important part of assessing the toxic potential of nanoparticles for specific applications should be the direct comparison of biological activities with those of alternative materials for the same application. Nanoparticulate inorganic ultraviolet (UV) filters, such as zinc oxide (ZnO), are commonly incorporated into transparent sunscreen and cosmetic formulations. However, concerns have been raised about potential unwanted effects, despite their negligible skin penetration and inherent advantages over organic chemical UV-filters. To provide useful application-relevant assessments of their potential hazard with/without UVA co-exposure, we directly compared cytotoxic and immune response profiles of human THP-1 monocytic cells to ZnO nanoparticles (30 nm) with bulk ZnO particulates (200 nm) and five conventional organic chemical UV-filters - butylmethoxydibenzoylmethane (avobenzone), octylmethoxycinnamate, octylsalicylate, homosalate and 4-methylbenzylidene camphor. High exposure concentrations of both organic and particulate UV-filters were required to cause cytotoxicity in monocyte and macrophage cultures after 24 h. Co-exposure with UVA (6.7 J/cm(2)) did not alter cytotoxicity profiles. Particle surface area-based dose responses showed that ZnO NPs were better tolerated than bulk ZnO. Organic and particulate UV-filters increased apoptosis at similar doses. Only particulates increased the generation of reactive oxygen species. Interleukin-8 (IL-8) release was increased by all particulates, avobenzone, homosalate and octylsalicylate. IL-1ß release was only increased in macrophages by exposure to avobenzone and homosalate. In conclusion, direct effects were caused in monocytes and macrophages at similar concentrations of both organic UV-filters and ZnO nanoparticulates - indicating that their intrinsic cytotoxicity is similar. With their lower skin penetration, ZnO nanoparticles are expected to have lower bioactivity when used in sunscreens.

Investigating the immunomodulatory nature of zinc oxide nanoparticles at sub-cytotoxic levels in vitro and after intranasal instillation in vivo.

BACKGROUND: This study evaluates the time-dependent pro-inflammatory response of the model human lung epithelial cells (A549) to industrially relevant zinc oxide nanoparticles (ZnO NPs). In terms of toxicity, ZnO-NPs are categorised into the group of high toxicity nanomaterials. However information on pro-inflammatory potential of these NPs at sub-toxic concentrations is limited. Understanding how cellular defense mechanisms function in the presence of sub-cytotoxic concentrations of these NPs is vital. Moreover, there is an urgent need for additional in vivo studies addressing pulmonary toxicity due to accidental inhalation of ZnO NPs. RESULTS: Exposure to sub-cytotoxic ZnO NP concentrations (20 µg/mL) induced significant up-regulation of mRNA for the pro-inflammatory cytokine IL-8 and redox stress marker heme oxygenase-1, along with increased release of IL-8. The highest pro-inflammatory response was recorded after 4 to 6 hr exposure to ZnO NPs over a 24 hr period. Pre-treatment of A549 cells with the sulfhydryl antioxidant N-acetyl cysteine (at 5 mM) resulted in significant reduction of the up-regulation of inflammatory markers, confirming the role of reactive oxygen species in the observed immunomodulatory effects, independent of cytotoxicity. Furthermore, we report for the first time that, intranasal instillation of a single dose (5 mg/kg) of pristine or surfactant-dispersed ZnO NPs can cause pulmonary inflammation, already after 24 hr in a murine model. This was confirmed by up-regulation of eotaxin mRNA in the lung tissue and release of pro-inflammatory cytokines in the sera of mice exposed to ZnO NPs. CONCLUSION: Our study highlights that even at sub-cytotoxic doses ZnO NPs can stimulate a strong inflammatory and antioxidant response in A549 cells. ZnO NP mediated cytotoxicity may be the outcome of failure of cellular redox machinery to contain excessive ROS formation. Moreover exposure to a single but relatively high dose of ZnO NPs via intranasal instillation may provoke acute pulmonary inflammatory reactions in vivo.

Reducing ZnO nanoparticle cytotoxicity by surface modification.

Nanoparticulate zinc oxide (ZnO) is one of the most widely used engineered nanomaterials and its toxicology has gained considerable recent attention. A key aspect for controlling biological interactions at the nanoscale is understanding the relevant nanoparticle surface chemistry. In this study, we have determined the disposition of ZnO nanoparticles within human immune cells by measurement of total Zn, as well as the proportions of extra- and intracellular dissolved Zn as a function of dose and surface coating. From this mass balance, the intracellular soluble Zn levels showed little difference in regard to dose above a certain minimal level or to different surface coatings. PEGylation of ZnO NPs reduced their cytotoxicity as a result of decreased cellular uptake arising from a minimal protein corona. We conclude that the key role of the surface properties of ZnO NPs in controlling cytotoxicity is to regulate cellular nanoparticle uptake rather than altering either intracellular or extracellular Zn dissolution.

Comparison of UVA-induced ROS and sunscreen nanoparticle-generated ROS in human immune cells.

Oxidative damage to cells and tissues from free radicals induced by ultraviolet (UV) irradiation can be attenuated by sunscreen components, such as ZnO and TiO2 nanoparticles (NPs). Although it is known that reactive oxygen species (ROS) are generated by cells upon exposure to ZnO and TiO2 NPs, it is unknown to what extent the amount generated is altered with UV co-exposure. As it is a critical component for determining the relative risk of these NPs when used in sunscreen formulations, we have investigated ROS generation by these NPs in human THP-1 monocyte immune cells following UVA co-exposure. Whilst the applied UVA dose (6.7 J cm(-2)) did not alter cell viability after 24 h, it induced significant ROS production - causing a 7-fold increase in intracellular peroxide and 3.3-fold increase in mitochondrial superoxide levels after 1 h. However, co-exposure to NPs and UVA generated the same or less ROS than with UVA exposure alone, with the exception of anatase TiO2, which showed significantly increased levels. These findings indicate that ROS generation from nanosunscreens is, in most cases, an insignificant contributor to the overall risk associated with oxidative stress from UVA exposure itself.

Quantification of ZnO nanoparticle uptake, distribution, and dissolution within individual human macrophages.

The usefulness of zinc oxide (ZnO) nanoparticles has led to their wide distribution in consumer products, despite only a limited understanding of how this nanomaterial behaves within biological systems. From a nanotoxicological viewpoint the interaction(s) of ZnO nanoparticles with cells of the immune system is of specific interest, as these nanostructures are readily phagocytosed. In this study, rapid scanning X-ray fluorescence microscopy was used to assay the number ZnO nanoparticles associated with ∼1000 individual THP-1 monocyte-derived human macrophages. These data showed that nanoparticle-treated cells endured a 400% elevation in total Zn levels, 13-fold greater than the increase observed when incubated in the presence of an equitoxic concentration of ZnCl2. Even after excluding the contribution of internalized nanoparticles, Zn levels in nanoparticle treated cells were raised ∼200% above basal levels. As dissolution of ZnO nanoparticles is critical to their cytotoxic response, we utilized a strategy combining ion beam milling, X-ray fluorescence and scanning electron microscopy to directly probe the distribution and composition of ZnO nanoparticles throughout the cellular interior. This study demonstrated that correlative photon and ion beam imaging techniques can provide both high-resolution and statistically powerful information on the biology of metal oxide nanoparticles at the single-cell level. Our approach promises ready application to broader studies of phenomena at the interface of nanotechnology and biology.

Relating cytotoxicity, zinc ions, and reactive oxygen in ZnO nanoparticle-exposed human immune cells.

Although zinc oxide (ZnO) nanoparticles (NPs) have been widely formulated in sunscreens, the relationship between reactive oxygen species (ROS) generation induced by these particles, zinc ions, and cytotoxicity is not clearly understood. This study explores whether these factors can be accurately quantified and related. The study demonstrates a strong correlation between ZnO NP-induced cytotoxicity and free intracellular zinc concentration (R (2) = .945) in human immune cells, indicating a requirement for NP dissolution to precede cytotoxicity. In addition, although direct exposure to ZnO NPs was found to induce cytotoxicity at relatively high concentrations, indirect exposure (via dialysis) was not cytotoxic, even at extremely high concentrations, highlighting a requirement for NP-to-cell contact. Elevated levels of ROS present in NP-exposed cells also correlated to both cytotoxicity and intracellular free zinc. Although the addition of antioxidant was able to reduce ROS, cytotoxicity to ZnO NPs was unaffected, suggesting ROS may be, in part, a result of cytotoxicity rather than a causal factor. This study highlights both the requirement and role of intracellular dissolution of zinc nanomaterials to elicit a cytotoxic response. This response is only partially ROS dependent, and therefore, modification of NP uptake and their intracellular solubility are key components in modulating the bioactivity of ZnO NPs.

Formation of zinc-containing nanoparticles from Zn²âº ions in cell culture media: implications for the nanotoxicology of ZnO.

Zinc ions generate a range of poorly soluble Zn-containing nanoparticles when added to commonly used mammalian cell culture media. The formation of these nanoparticles confounds the use of soluble Zn salts as positive controls during cytotoxicity testing of other Zn-containing nanoparticles, such as ZnO. These nanoprecipitates can either be crystalline or amorphous and vary in composition depending upon the concentration of Zn(II) within the medium. The cytotoxicity and immune system response of these nanoparticles in situ are similar to those of 30 nm ZnO nanoparticles. The low residual level of truly soluble Zn species (taken as species passing through a 2 kDa membrane) in cell culture media with serum is insufficient to elicit any appreciable cytotoxicity. These observations highlight the importance of employing appropriate controls when studying ZnO nanoparticle toxicity and suggest a re-evaluation of the conclusions drawn in some previous cytotoxicity studies.

Independent cytotoxic and inflammatory responses to zinc oxide nanoparticles in human monocytes and macrophages.

Significant public and scientific concerns remain for the use of nanoparticles (NPs) in commercial products, particularly those applied topically for skin care. There are currently a range of metal oxides formulated into many sunscreens that are present at the nanoscale. In this study, we sought to determine the effect of the size and dispersion of one type of these NPs (zinc oxide) on immune cell function and cytotoxicity for human macrophages and monocytes, which are key cells for particle and debris clearance in the skin. We have found that particle size and coating, but surprisingly, not agglomeration, are key determinates of nanoparticle cytotoxicity in an in vitro culture system of human immune cells. Most importantly, we found that this nanoparticle-induced cellular immune signalling, can be decoupled from cytotoxicity and surface coating, so that at an equivalent cytotoxic load, smaller particles induce a greater cellular response.

Increased vascular endothelial growth factor and receptors: relationship to angiogenesis in asthma.

RATIONALE: Increased vascularity is a feature of airway remodeling in asthma with the potential to contribute to a number of functional abnormalities in this chronic disease. Although various growth factors have been implicated in modulating vascularity, the important contributors in vivo are still being elucidated. The most likely candidate is vascular endothelial growth factor (VEGF). OBJECTIVES: We have examined VEGF and its receptors, VEGFR1 and VEGFR2, and angiopoietin-1 (Ang1) in the airways of subjects with asthma and contrasted these results with findings in normal control subjects. We aimed to explore whether these powerful angiogenic factors were expressed at elevated levels in asthmatic airways. METHODS: We obtained biopsy and bronchoalveolar lavage samples from 35 subjects with mild to moderate asthma and from 22 normal control subjects. MEASUREMENTS: We performed immunohistochemistry and image analysis to obtain quantitative measures of VEGF, VEGFR1, VEGFR2, and Ang1 staining in airway biopsies, and ELISA to assess VEGF concentration in the bronchoalveolar lavage fluid. RESULTS: VEGF staining and VEGF levels in bronchoalveolar lavage fluid were elevated in the airways of subjects with asthma and were related to the number of vessels; Ang1 staining was similarly increased. VEGFR1 was slightly higher in subjects with asthma and the VEGFR1:VEGFR2 ratio was significantly higher in subjects with asthma. We observed angiogenic sprouts (i.e., early-forming vascular structures) that were increased in number in subjects with asthma. CONCLUSIONS: Our findings suggest that VEGF, its receptors, and Ang1 are likely to be important in vascular changes in the airways of patients with asthma. Further, there are observable structures in the vessel walls of asthmatic airways that could present ongoing evidence of increased angiogenic activity.

BAL eotaxin and IL-5 in asthma, and the effects of inhaled corticosteroid and beta2 agonist.

OBJECTIVE: In a longitudinal ex vivo placebo-controlled study, asthmatics already treated with inhaled corticosteroid received supplemental long-acting beta-agonist (LABA) or increased doses of their inhaled corticosteroid (ICS). Previously reports have shown significant reductions in biopsy eosinophil numbers after treatment with LABA. Following these findings, eosinophil chemokines eotaxin and IL-5 in the BAL fluid at baseline and after 3 months of study medication have now been measured, and these data with that from new cross-sectional controls have also been compared. It is hypothesised that changes in airway eosinophils would be related to eosinophil cytokines. METHODOLOGY: BAL cytokines were measured by chemiluminescent enzyme-linked immunosorbent assay, while eosinophils were measured by immunohistochemistry or differential cell counting. For all measures, longitudinal data were compared to that from ICS-free asthmatics (n = 42) and non-asthmatic controls (n = 28). RESULTS: BAL eotaxin in asthmatics was elevated above non-asthmatic levels regardless of ICS levels. BAL IL-5 was elevated in ICS-free asthmatics, but not in asthmatics on low-dose ICS treatment. Longitudinally, BAL eotaxin was unchanged after 3 months. Unexpectedly, IL-5 increased after 3 months of additional LABA treatment but was not further affected by increasing the dose of ICS. Airway eotaxin seemed to be constitutively raised in asthmatics, whereas, IL-5 levels were more steroid-responsive. No relationship was observed between eosinophils and eosinophilic cytokines in the BAL. CONCLUSIONS: While the elevation of luminal IL-5 with LABA treatment cannot be accounted for, it may have contributed to luminal clearance of airway wall eosinophils. The lack of correlation between airways eosinophils and eosinophilic cytokines in the BAL is particularly important.