Gold Nanorods as Radiopharmaceutical Carriers: Preparation and Preliminary Radiobiological In Vitro Tests.

Low-energy electrons (Auger electrons) can be produced via the interaction of photons with gold atoms in gold nanorods (AuNRs). These electrons are similar to those emitted during the decay of technetium-99m (99mTc), a radioactive nuclide widely used for diagnostics in nuclear medicine. Auger and internal conversion (IC) electron emitters appropriately targeted to the DNA of tumors cells may, therefore, represent a new radiotherapeutic approach. 99mTc radiopharmaceuticals, which are used for diagnosis, could indeed be used in theragnostic fields when loaded on AuNRs and delivered to a tumor site. This work aims to provide a proof of concept (i) to evaluate AuNRs as carriers of 99mTc-based radiopharmaceuticals, and (ii) to evaluate the efficacy of Auger electrons emitted by photon-irradiated AuNRs in inducing radio-induced damage in T98G cells, thus mimicking the effect of Auger electrons emitted during the decay of 99mTc used in clinical settings. Data are presented on AuNRs' chemical characterization (with an aspect ratio of 3.2 and Surface Plasmon Resonance bands at 520 and 680 nm) and the loading of pharmaceuticals (after 99mTc decay) on their surface. Spectroscopic characterizations, such as UV-Vis and synchrotron radiation-induced X-ray photoelectron (SR-XPS) spectroscopies, were performed to investigate the drug-AuNR interaction. Finally, preliminary radiobiological data on cell killing with AuNRs are presented.

In Vitro Antiviral and Anti-Inflammatory Activities of N-Acetylglucosamine: Development of an Alternative and Safe Approach to Fight Viral Respiratory Infections.

Viral respiratory tract infections (RTIs) are responsible for significant morbidity and mortality worldwide. A prominent feature of severe respiratory infections, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is the cytokine release syndrome. Therefore, there is an urgent need to develop different approaches both against viral replication and against the consequent inflammation. N-acetylglucosamine (GlcNAc), a glucosamine (GlcN) derivative, has been developed as an immunomodulatory and anti-inflammatory inexpensive and non-toxic drug for non-communicable disease treatment and/or prevention. Recent studies have suggested that GlcN, due to its anti-inflammatory activity, could be potentially useful for the control of respiratory virus infections. Our present study aimed to evaluate in two different immortalized cell lines whether GlcNAc could inhibit or reduce both viral infectivity and the inflammatory response to viral infection. Two different viruses, frequent cause of upper and lower respiratory tract infections, were used: the H1N1 Influenza A virus (IAV) (as model of enveloped RNA virus) and the Human adenovirus type 2 (Adv) (as model of naked DNA virus). Two forms of GlcNAc have been considered, bulk GlcNAc and GlcNAc in nanoform to overcome the possible pharmacokinetic limitations of GlcNAc. Our study suggests that GlcNAc restricts IAV replication but not Adv infection, whereas nano-GlcNAc inhibits both viruses. Moreover, GlcNAc and mainly its nanoformulation were able to reduce the pro-inflammatory cytokine secretion stimulated by viral infection. The correlation between inflammatory and infection inhibition is discussed.

Nanoparticle Impact on the Bacterial Adaptation: Focus on Nano-Titania.

Titanium dioxide nanoparticles (nano-titania/TiO2 NPs) are used in different fields and applications. However, the release of TiO2 NPs into the environment has raised concerns about their biosafety and biosecurity. In light of the evidence that TiO2 NPs could be used to counteract antibiotic resistance, they have been investigated for their antibacterial activity. Studies reported so far indicate a good performance of TiO2 NPs against bacteria, alone or in combination with antibiotics. However, bacteria are able to invoke multiple response mechanisms in an attempt to adapt to TiO2 NPs. Bacterial adaption arises from global changes in metabolic pathways via the modulation of regulatory networks and can be related to single-cell or multicellular communities. This review describes how the impact of TiO2 NPs on bacteria leads to several changes in microorganisms, mainly during long-term exposure, that can evolve towards adaptation and/or increased virulence. Strategies employed by bacteria to cope with TiO2 NPs suggest that their use as an antibacterial agent has still to be extensively investigated from the point of view of the risk of adaptation, to prevent the development of resistance. At the same time, possible effects on increased virulence following bacterial target modifications by TiO2 NPs on cells or tissues have to be considered.

Exposure to TiO2 Nanoparticles Increases Listeria monocytogenes Infection of Intestinal Epithelial Cells.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in a variety of consumer products. Cellular exposure to TiO2 NPs results in complex effects on cell physiology that could impact biological systems. We investigated the behavior of Listeria monocytogenes in intestinal epithelial cells pre-treated with either a low or high (1 and 20 µg/cm2) dose of TiO2 NPs. Our results indicate that the pre-treated cells with a low dose became more permissive to listeria infection; indeed, both adhesion and invasion were significantly increased compared to control. Increased invasion seems to be correlated to cytoskeletal alterations induced by nanoparticles, and higher bacterial survival might be due to the high levels of listeriolysin O that protects L. monocytogenes from reactive oxygen species (ROS). The potential risk of increased susceptibility to L. monocytogenes infection related to long-term intake of nanosized TiO2 at low doses should be considered.

Use of a common European approach for nanomaterials' testing to support regulation: a case study on titanium and silicon dioxide representative nanomaterials.

The European Union (EU) continuously takes ensuring the safe use of manufactured nanomaterials (MNMs) in consumer products into consideration. The application of a common approach for testing MNMs, including the use of optimized protocols and methods' selection, becomes increasingly important to obtain reliable and comparable results supporting the regulatory framework. In the present study, we tested four representative MNMs, two titanium dioxides (NM100 and NM101) and two silicon dioxides (NM200 and NM203), using the EU FP7-NANoREG approach, starting from suspension and dispersion preparations, through to their characterization and final evaluation of biological effects. MNM dispersions were prepared following a refined NANOGENOTOX protocol and characterized by dynamic light scattering (DLS) in water/bovine serum albumin and in media used for in vitro testing. Potential genotoxic effects were evaluated on human bronchial BEAS-2B cells using micronucleus and Comet assays, and pro-inflammatory effects by cytokines release. Murine macrophages RAW 264.7 were used to detect potential innate immune responses using two functional endpoints (pro-inflammatory cytokines and nitric oxide [NO] production). The interaction of MNMs with RAW 264.7 cells was studied by electron microscopy. No chromosomal damage and slight DNA damage and an oxidative effect, depending on MNMs, were observed in bronchial cells. In murine macrophages, the four MNMs directly induced tumor necrosis factor α or interleukin 6 secretion, although at very low levels; lipopolysaccharide-induced NO production was significantly decreased by the titania and one silica MNM. The application of this approach for the evaluation of MNM biological effects could be useful for both regulators and industries.

Critical issues in genotoxicity assessment of TiO2 nanoparticles by human peripheral blood mononuclear cells.

In the last years, a number of in vitro studies have been performed to assess the genotoxic activity of titanium dioxide (TiO2 ). To resolve the contradictory results, in this study, we investigated the genotoxic activity of commercial TiO2 nanoparticles (NPs) and microparticles of different forms (anatase, rutile and mix of both). We evaluated micronucleus formation in stimulated lymphocytes, as well as DNA strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine in peripheral blood mononuclear cells (PBMCs), a mixed population of lymphocytes and monocytes. Different responses to TiO2 exposure were obtained depending on the assay. Both TiO2 NPs and microparticles and all the crystalline forms elicited a significant increase in 8-oxo-7,8-dihydro-2'-deoxyguanosine and DNA strand breaks in the whole PBMC population, without a concurrent increase of micronuclei in proliferating lymphocytes. The distribution of DNA damage in PBMCs, detected by the comet assay, that measures DNA damage at level of single cells, indicated the presence of a more susceptible cell subpopulation. The measurement of side scatter signals by flow cytometry highlighted the preferential physical interaction of TiO2 particles with monocytes that also displayed higher reactive oxygen species generation, providing a mechanistic explanation for the different responses observed in genotoxicity assays with PBMCs and lymphocytes. This study confirmed the suitability of human PBMCs as multi-cell model to investigate NP-induced DNA damage, but suggested some caution in the use of stimulated lymphocytes for the assessment of NP clastogenicity.

Analysis of major pollutants and physico-chemical characteristics of PM2.5 at an urban site in Rome.

Air quality data from a one year study at an urban roadside location in Rome are reported for major pollutants. Continuous concentration data of carbon monoxide, ozone, nitrogen dioxide, aromatic hydrocarbons and natural radioactivity were measured in the urban air of Rome from January 2016 to January 2017. Moreover, PM2.5 mass concentration and physico-chemical characteristics of single constituent particles are herein reported. Gaseous pollutants, except ozone, and PM2.5 showed maximum concentrations in December due to high atmospheric stability. O3 and NO2 trend analysis showed photochemical smog episodes in June and September. In September, during a photochemical smog episode the aromatic hydrocarbons contribution to ozone formation was experimentally proven. Pearson's coefficient among aromatic hydrocarbons and the ratio Toluene/Benzene (T/B) showed that pollutants were under the influence of vehicular traffic. Physico-chemical characterization of PM2.5 single particles, carried out by field emission scanning electron microscope combined with energy dispersive X-ray spectroscopy, displayed the presence of particle diversity from natural and anthropogenic origin. Four principal components in the PM2.5 were identified: carbonaceous particles, Ca-sulphates, soil dust and building structure particles, metal particles. The principal source of carbonaceous particles in this urban area consists of the motor vehicle exhausts and the heating systems in winter. Traces of S and sometimes S, Na, K were detected on varying percentages of carbonaceous particles. These data suggested that the carbonaceous particles act as vehicles for strong acids, prevalently H2SO4 and alkaline metal sulphates. A Saharan dust contribution to PM2.5 was found in different periods. Metal particles included iron oxide particles, metals oxide particles and Fe-rich metal compounds. The identification of chemical composition of individual particles provide useful information to determine their origin and formation processes.

Short-term oral exposure to low doses of nano-sized TiO2 and potential modulatory effects on intestinal cells.

The present study investigated potential modulatory effects of low doses of nano-sized titanium dioxide (TiO2) on intestinal cells in vivo and in vitro. After short-term exposure to TiO2 nanoparticles in rats, histopathological analysis of intestinal tissues indicated a gender-specific effect with increased length of intestinal villi in male rats only. Moreover the intestinal tissue showed nanoparticle deposition as revealed by ICP-MS determination of titanium. Increased serum testosterone levels were also detected. Considering the male-specific effects detected in vivo, the TiO2 nanoparticle interaction with intestinal cells was further characterized in vitro and the modulating effect of testosterone and a hormone-induced growth factor, namely Insulin-like Growth Factor 1 (IGF-1), was also assessed. Cytotoxicity assays and analysis of Reactive Oxygen Species (ROS) production showed neither cellular alteration nor oxidative stress for nanoparticles at low concentrations, even though they were able to penetrate intestinal cells, as revealed by electron microscopy. Cell treatments with nanoparticles in association with testosterone or IGF-1 showed increased cell proliferation, compared to nanoparticles or testosterone/IGF-1 alone. Since long-term intake of TiO2 nanoparticles at low doses is a relevant scenario for human exposure, attention should be given to the potential modulating activity of this nanomaterial on cell proliferation.

ZnO nanoparticle tracking from uptake to genotoxic damage in human colon carcinoma cells.

Zinc Oxide (ZnO) nanoparticles are widely used both in the industry and in biomedical applications for their chemical and physical nanomaterial properties. It is therefore essential to go in depth into the cytotoxicity mechanisms and interactions between nanomaterials and cells. The aim of this work was to evaluate the dissolution of ZnO nanoparticles and their uptake, from a few minutes after treatments up to 24h. ZnO nanoparticles routes of entry into the human colon carcinoma cells (LoVo) were followed at different times by a thorough ultrastructural investigation and semiquantitative analysis. The intracellular release of Zn(2+) ions by Zinquin fluorescent dye, and phosphorylated histone H2AX (γ-H2AX) expression were evaluated. The genotoxic potential of ZnO nanoparticles was also investigated by determining the levels of 8-hydroxyl-2'-deoxyguanosine (8-oxodG). The experimental data show that ZnO nanoparticles entered LoVo cells by either passive diffusion or endocytosis or both, depending on the agglomeration state of the nanomaterial. ZnO nanoparticles coming into contact with acid pH of lysosomes altered organelles structure, resulting in the release of Zn(2+) ions. The simultaneous presence of ZnO nanoparticles and Zn(2+) ions in the LoVo cells determined the formation of reactive oxygen species at the mitochondrial and nuclear level, inducing severe DNA damage.

Different mechanisms are involved in oxidative DNA damage and genotoxicity induction by ZnO and TiO2 nanoparticles in human colon carcinoma cells.

In this work we investigated the genotoxicity of zinc oxide and titanium dioxide nanoparticles (ZnO NPs; TiO2 NPs) induced by oxidative stress on human colon carcinoma cells (Caco-2 cells). We measured free radical production in acellular conditions by Electron Paramagnetic Resonance technique and genotoxicity by micronucleus and Comet assays. Oxidative DNA damage was assessed by modified Comet assay and by measuring 8-oxodG steady state levels. The repair kinetics of DNA oxidation as well as the expression levels of hOGG1 were also analyzed. Even if both NPs were able to produce ROS in acellular conditions and to increase 8-oxodG levels in Caco-2 cells, only ZnO NPs resulted genotoxic inducing micronuclei and DNA damage. Furthermore, Caco-2 cells exposed to ZnO NPs were not able to repair the oxidative DNA damage that was efficiently repaired after TiO2 NPs treatment, through OGG1 involvement. These results indicate that the high oxidant environment caused by ZnO NPs in our cellular model can induce DNA damage and affect the repair pathways.

Listeria monocytogenes behaviour in presence of non-UV-irradiated titanium dioxide nanoparticles.

Listeria monocytogenes is the agent of listeriosis, a food-borne disease. It represents a serious problem for the food industry because of its environmental persistence mainly due to its ability to form biofilm on a variety of surfaces. Microrganisms attached on the surfaces are a potential source of contamination for environment and animals and humans. Titanium dioxide nanoparticles (TiO2 NPs) are used in food industry in a variety of products and it was reported that daily exposure to these nanomaterials is very high. Anti-listerial activity of TiO2 NPs was investigated only with UV-irradiated nanomaterials, based on generation of reactive oxigen species (ROS) with antibacterial effect after UV exposure. Since both Listeria monocytogenes and TiO2 NPs are veicolated with foods, this study explores the interaction between Listeria monocytogenes and non UV-irradiated TiO2 NPs, with special focus on biofilm formation and intestinal cell interaction. Scanning electron microscopy and quantitative measurements of biofilm mass indicate that NPs influence both production and structural architecture of listerial biofilm. Moreover, TiO2 NPs show to interfere with bacterial interaction to intestinal cells. Increased biofilm production due to TiO2 NPs exposure may favour bacterial survival in environment and its transmission to animal and human hosts.

Oral, short-term exposure to titanium dioxide nanoparticles in Sprague-Dawley rat: focus on reproductive and endocrine systems and spleen.

The study explored possible reproductive and endocrine effects of short-term (5 days) oral exposure to anatase TiO2 nanoparticles (0, 1, 2 mg/kg body weight per day) in rat. Nanoparticles were characterised by scanning electron microscopy (SEM) and transmission electron microscopy, and their presence in spleen, a target organ for bioaccumulation, was investigated by single-particle inductively coupled plasma mass spectrometry and SEM/energy-dispersive X-ray. Analyses included serum hormone levels (testosterone, 17-ß-estradiol and triiodothyronine) and histopathology of thyroid, adrenals, ovary, uterus, testis and spleen. Increased total Ti tissue levels were found in spleen and ovaries. Sex-related histological alterations were observed at both dose levels in thyroid, adrenal medulla, adrenal cortex (females) and ovarian granulosa, without general toxicity. Altered thyroid function was indicated by reduced T3 (males). Testosterone levels increased in high-dose males and decreased in females. In the spleen of treated animals TiO2 aggregates and increased white pulp (high-dose females) were detected, even though Ti tissue levels remained low reflecting the low doses and the short exposure time. Our findings prompt to comprehensively assess endocrine and reproductive effects in the safety evaluation of nanomaterials.

Comparative study of ZnO and TiO2 nanoparticles: physicochemical characterisation and toxicological effects on human colon carcinoma cells.

Despite human gastrointestinal exposure to nanoparticles (NPs), data on NPs toxicity in intestinal cells are quite scanty. In this study we evaluated the toxicity induced by zinc oxide (ZnO) and titanium dioxide (TiO2) NPs on Caco-2 cells. Only ZnO NPs produced significant cytotoxicity, evaluated by two different assays. The presence of foetal calf serum in culture medium significantly reduced ZnO NPs toxicity as well as ion leakage and NP-cell interaction. The two NPs increased the intracellular amount of reactive oxygen species (ROS) after 6 h treatment. However, only ZnO NPs increased ROS and induced IL-8 release both after 6 and 24 h. Experimental data indicate a main role of chemical composition and solubility in ZnO NPs toxicity. Moreover our results suggest a key role of oxidative stress in ZnO NPs cytotoxicity induction related both to ion leakage and to cell interaction with NPs in serum-free medium.

Exposure to ZnO nanoparticles induces oxidative stress and cytotoxicity in human colon carcinoma cells.

Engineered nanoparticles offer great promise in many industrial and biomedical applications, however little information is available about gastrointestinal toxicity. The purpose of this study was to assess the cytotoxicity, oxidative stress, apoptosis and proinflammatory mediator release induced by ZnO nanoparticles on human colon carcinoma LoVo cells. The biological activity of these particles was related to their physico-chemical characteristics. The physico-chemical characteristics were evaluated by analytical electron microscopy. The cytotoxicity was determined by growth curves and water-soluble tetrazolium assay. The reactive oxygen species production, cellular glutathione content, changes of mitochondrial membrane potential and apoptosis cell death were quantified by flow cytometry. The inflammatory cytokines were evaluated by enzyme-linked immunoadsorbent assay. Treatment with ZnO (5µg/cm(2) corresponding to 11.5µg/ml) for 24h induced on LoVo cells a significant decrease of cell viability, H2O2/OH increase, O2(-) and GSH decrease, depolarization of inner mitochondrial membranes, apoptosis and IL-8 release. Higher doses induced about 98% of cytotoxicity already after 24h of treatment. The experimental data show that oxidative stress may be a key route in inducing the cytotoxicity of ZnO nanoparticles in colon carcinoma cells. Moreover, the study of the relationship between toxicological effects and physico-chemical characteristics of particles suggests that surface area does not play a primary role in the cytotoxicity.

[The "silica" component in the PM10 of an urban site]. / La componente "silice" nel PM10 di un ambiente urbano.

BACKGROUND: In vivo and in vitro toxicological studies have shown that the aged fracturated crystalline silica, which is a component of airborne particulate, exerts an important inflammatory action on airways. The evaluation of the concentration level of airborne crystalline silica in an urban area is an important research subject in order to determine the exposure levels of the general population. OBJECTIVES: The aim was to study the seasonal trend of the quartz (the most common form of crystalline silica) concentration levels in the particulate inhalable faction (PM10) in the urban area of Rome. METHODS: PM10, sampled by a cascade impactor, was analysed by scanning electron microscopy, equipped with a thin-window system for X-ray microanalysis (SEM/EDX) for qualitative analysis. Parallely the concentration levels of quartz in the particulate were determined by X-ray diffractometry (XRD) for quantitative analysis, using the NIOSH 7500 method (NIOSH, 1994). RESULTS: From September 2004 to October 2005 the abundance of silica particles, evaluated by SEM/EDX was in the range 1.6/10.4%, with a concentration level of free crystalline silica in the range 0.25/2.87 microg/mi. The equivalent diameter of silica particles ranged from 0.3 to 10.5 mircom, moreover, more than 87% of particles showed a diameter less than 2.5 microm. CONCLUSIONS: The correlations between SEM/EDX and XRD data seem to suggest that the airborne silica particles in the urban location studied consisted mostly of crystalline silica. Moreover, the data suggest the existence of a significant contribution of silica particles due to southwest wind carrying a fine dust from the Sahara desert to Mediterranean Europe.

Evaluation of emission toxicity of urban bus engines: compressed natural gas and comparison with liquid fuels.

Emissions from a spark-ignition (SI) heavy-duty (HD) urban bus engine with a three-way catalyst (TWC), fuelled with compressed natural gas (CNG), were chemically analyzed and tested for genotoxicity. The results were compared with those obtained in a previous study on an equivalent diesel engine, fuelled with diesel oil (D) and a blend of the same with 20% vegetable oil (B20). Experimental procedures were identical, so that emission levels of the CNG engine were exactly comparable to the ones of the diesel engine. The experimental design was focused on carcinogenic compounds and genotoxic activity of exhausts. The results obtained show that the SI CNG engine emissions, with respect to the diesel engine fuelled with D, were nearly 50 times lower for carcinogenic polycyclic aromatic hydrocarbons (PAHs), 20 times lower for formaldehyde, and more than 30 times lower for particulate matter (PM). A 20-30 fold reduction of genotoxic activity was estimated from tests performed. A very high reduction of nitrogen oxides (NO(X)) was also measured. The impact of diesel powered transport on urban air quality, and the potential benefits deriving from the use of CNG for public transport, are discussed.

Winter urban air particles from Rome (Italy): effects on the monocytic-macrophagic RAW 264.7 cell line.

Epidemiological data show an association between exposure to elevated levels of particulate matter (PM), in particular the fine fraction (<2.5 microm in diameter), and an increase in cardiovascular mortality and respiratory symptoms. The aim of this study was to compare the in vitro toxicity of coarse and fine particulate matter collected with a cascade impactor during winter in an urban area of Rome in relation to their physicochemical characterization (size distribution and chemical composition) as assessed by analytical electron microscopy (SEM/EDX). The X-ray microanalysis data of single particles of coarse and fine matter were analyzed by hierarchical cluster analysis to determine the principal component of the two granulometric fractions. The main chemical difference between the two fractions was the greater abundance of carbonaceous particles in the fine fraction. We compared the ability of coarse and fine fractions, carbon black (CB), and residual oil fly ash (ROFA) to induce arachidonic acid release and tumor necrosis factor-alpha (TNF-alpha) production in the monocytic-macrophagic RAW 264.7 cell line at concentrations of 30 and 120 microg/mL. Our results showed that CB and ROFA were consistently less effective than both fractions of urban particles at inducing an inflammatory reaction in RAW 264.7 cells. Both PM fractions dose-dependently increased TNF-alpha production in RAW 264.7 cells after 5 and 24h of incubation, and only the TNF-alpha production induced by coarse particles at 30 microg/mL decreased significantly (P<0.01) after 24h of treatment. In our in vitro model the winter fine fraction was more reactive than the winter coarse fraction, in contrast to a previously examined summer sample. In the summer sample, coarse particles produced higher levels of inflammatory mediators than fine particles and the CB was consistently less effective than the urban particles. The different behaviors between summer and winter urban fractions may be due to their different physicochemical characteristics; in fact, the comparison of the two samples' characterization by SEM/EDX and X-ray photoelectron spectroscopy (XPS) analysis showed that in winter the carbonaceous particles are more abundant than in summer and that winter particles carry a greater quantity of organic compounds. We suggest that the higher concentration of organic compounds on fine carbonaceous particles may partially explain the higher activation of RAW 264.7 cells by fine particles.

Emission comparison of urban bus engine fueled with diesel oil and 'biodiesel' blend.

The chemical and toxicological characteristics of emissions from an urban bus engine fueled with diesel and biodiesel blend were studied. Exhaust gases were produced by a turbocharged EURO 2 heavy-duty diesel engine, operating in steady-state conditions on the European test 13 mode cycle (ECE R49). Regulated and unregulated pollutants, such as carcinogenic polycyclic aromatic hydrocarbons (PAHs) and nitrated derivatives (nitro-PAHs), carbonyl compounds and light aromatic hydrocarbons were quantified. Mutagenicity of the emissions was evaluated by the Salmonella typhimurium/mammalian microsome assay. The effect of the fuels under study on the size distribution of particulate matter (PM) was also evaluated. The use of biodiesel blend seems to result in small reductions of emissions of most of the aromatic and polyaromatic compounds; these differences, however, have no statistical significance at 95% confidence level. Formaldehyde, on the other hand, has a statistically significant increase of 18% with biodiesel blend. In vitro toxicological assays show an overall similar mutagenic potency and genotoxic profile for diesel and biodiesel blend emissions. The electron microscopy analysis indicates that PM for both fuels has the same chemical composition, morphology, shape and granulometric spectrum, with most of the particles in the range 0.06-0.3 microm.

Fine environmental particulate engenders alterations in human lung epithelial A549 cells.

Particulate matter (PM), a component of urban air pollution that derives primarily from the combustion of fossil fuels, is responsible for a number of health effects in humans. Recent studies have demonstrated that the fine particles (PM(2.5)) present in high numbers in PM samples can be more harmful than larger particles, since they are more efficiently retained in the peripheral lung. In the present study, we have investigated the biological effects of PM(2.5) on human lung epithelial cell line A549. Morphological analysis performed by immunofluorescence and electron microscopy showed that fine particles interact with the cell surface, where they induce evident alterations and, subsequently, are internalized in the cytoplasm. Cytoskeletal components, in particular microfilaments and microtubules, cause modifications upon challenge with PM(2.5). Of interest, an early cell response to the fine particulate is an increase of reactive oxygen species content, which can account for the observed cytoskeletal changes and the production of proinflammatory cytokines in A549 cells. In particular, exposure to PM(2.5) promoted a dose- and time-dependent release of TNF-alpha and IL-6 in the cell medium.

Environmental fine particulate matter (PM 2.5) activates the RAW 264.7 macrophage cell line even at very low concentrations as revealed by 1H NMR.

Because of the association between inhalation of airborne particulate matter (PM) and human respiratory and cardiovascular disease, it is necessary to understand the tissue damage induced by these particles. One of the cell types principally involved in the body's reaction to PM are macrophages, which remove particles in the airway passages and the lungs through phagocytosis. In fact, when macrophages are exposed to a toxic agent such as PM, they undergo a series of changes (including variations in morphology, an increase in glycolysis, and consequent lactate production and the release of cytokines such as interleukin-6 and tumor necrosis factor-alpha) necessary to transform them from "resting" to "activated" macrophages. Because (1)H NMR is extremely useful in monitoring, noninvasively, macrophage metabolism and because this technique has never been utilized to examine macrophage activation after exposure to PM, it was the purpose of the present study to investigate the effects of PM exposure on the RAW 264.7 stabilized macrophage cell line using (1)H NMR spectroscopy. PM with a diameter <2.5 microm (PM 2.5) was utilized because a closer association to mortality and adverse respiratory health effects has been found with this fraction than with particles of a larger size. Measurements were conducted on whole cells at both 500 and 700 MHz as well as on perchloric acid extracts at 700 MHz. Significant variations in numerous metabolites were seen at very low concentrations of PM 2.5. Many of these changes point to activation of RAW 264.7 macrophages even at doses of PM 2.5 much lower than those commonly employed in cell studies. These results are particularly significant since the same concentrations of PM did not induce changes in morphology and release of cytokines in these cells. Therefore, (1)H NMR spectroscopy is an extremely sensitive probe in observing subtle variations in macrophages after exposure to PM 2.5.