Human Lung Cell Line A549 Research Articles

Cytotoxic effects of crystalline silica in form of micro and nanoparticles on the human lung cell line A549.

Airborne crystalline silica (SiO2) particles are one of the most common pollutants in stone industries. Limited studies have investigated the health effects of crystalline SiO2 nanoparticles. Hence, the objective of this study was to study the cytotoxicity of SiO2 in nano and micron sizes. A mineral quartz sample in the range of 0.2-0.8mm sizes was purchased. These particles were ground at about 5 and 0.1 microns. Human cell line A549 was exposed to micro and nanometer particles at concentrations of 10, 50, 100, and 250μg/ml for 24 and 72 h. Subsequently, the cytotoxicity of exposed cells was investigated by measuring cell survival, ROS generation, mitochondrial permeability, and intracellular glutathione content. The results showed that crystalline SiO2 nano and microparticles decreased cell survival, increased ROS generation, damaged the mitochondrial membrane, and lowered the antioxidant content of these cells in a concentration- and time-dependent manner. The toxicity of crystalline SiO2 microparticles at concentrations ≤50μg/mL was greater than for nanoparticles, which was the opposite at concentrations ≥100μg/mL. Exposure time and concentration were crucial factors for the cytotoxicity of exposed A549 cells to crystalline SiO2 particles, which can affect the severity of the effect of particle size. Due to the limitation of exposure concentration and test durations in this study, further studies on the parameters of nanoparticle toxicity and underlying mechanisms could advance our knowledge.

Study on the cytotoxic, antimetastatic and albumin binding properties of the oxidovanadium(IV) chrysin complex. Structural elucidation by computational methodologies

We have previously synthesized and characterized the chrysin coordination complex with the oxidovanadium(IV) cation (VIVO(chrys)2) and characterized in ethanolic solution and in solid state. Because suitable single crystals for X-ray diffraction determinations could not be obtained, in the present work, we elucidate the geometrical parameters of this complex by computational methodologies. The optimization and vibrational investigation were carried out both in ethanolic solution and in gas phase. The computational results support the experimentally proposed geometries of the VIVO(chrys)2 complex, thus leading to the conclusion that the complex exists as conformers with trans-octahedral geometry in ethanolic solution and as conformers with cis-octahedral geometry in the solid state. The complex also exists as conformers with trans-octahedral geometry in aqueous media. The active species formed after dissolution in DMSO showed anticancer and antimetastatic behavior in human lung cell line A549 with moderate binding (Kaca. 105 M−1) to bovine serum albumin (BSA). The interaction through hydrogen bonding and van der Waals forces resulted in a spontaneous process. Site marker competitive experiments showed binding sites for chrysin mainly located in site II (subdomain IIIA) and in site I (subdomain IIIA) for the complex. FT-IR spectral measurements showed evidences of the alterations of protein secondary structure in the presence of chrysin and VIVO(chrys)2.

A stealth antigen SPESP1, which is epigenetically silenced in tumors, is a suitable target for cancer immunotherapy.

Recent studies have revealed that tumor cells decrease their immunogenicity by epigenetically repressing the expression of highly immunogenic antigens to survive in immunocompetent hosts. We hypothesized that these epigenetically hidden “stealth” antigens should be favorable targets for cancer immunotherapy due to their high immunogenicity. To identify these stealth antigens, we treated human lung cell line A549 with DNA methyltransferase inhibitor 5‐aza‐2′‐deoxycytidine (5Aza) and its prodrug guadecitabine for 3 d in vitro and screened it using cDNA microarray analysis. We found that the gene encoding sperm equatorial segment protein 1 (SPESP1) was re‐expressed in cell lines including solid tumors and leukemias treated with 5Aza, although SPESP1 was not detected in untreated tumor cell lines. Using normal human tissue cDNA panels, we demonstrated that SPESP1 was not detected in normal human tissue except for testis and placenta. Moreover, we found using immunohistochemistry SPESP1 re‐expression in xenografts in BALB/c‐nu/nu mice that received 5Aza treatment. To assess the antigenicity of SPESP1, we stimulated human CD4+ T‐cells with a SPESP1‐derived peptide designed using a computer algorithm. After repetitive stimulation, SPESP1‐specific helper T‐cells were obtained; these cells produced interferon‐γ against HLA‐matched tumor cell lines treated with 5Aza. We also detected SPESP1 expression in freshly collected tumor cells derived from patients with acute myeloid leukemia or lung cancer. In conclusion, SPESP1 can be classified as a stealth antigen, a molecule encoded by a gene that is epigenetically silenced in tumor cells but serves as a highly immunogenic antigen suitable for cancer immunotherapy.

Phytochemical Biological and Cytotoxic Activities of Calothamnus quadrifidus Leaves Cultivated in Egypt

The hydro distilled essential oil obtained from Calothamnus quadrifidus leaves was analyzed by gas liquid chromatography-mass spectroscopy. Quantification of the oil constituents was carried out using the gas liquid chromatography-flame ionization detection device where 1,8-cineole was quantified as 525.62 μg/ml. The oil showed potential antioxidant activity when tested by 1,1-diphenyl-2-picrylhydrazine radical scavenging assay where the concentration required to scavenge 1,1-diphenyl-2-picrylhydrazine by 50 %=37.3 μg. The cytotoxic activity of the essential oil was evaluated using 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay compared with 1,8-cineole and doxorubicin. Calothamnus quadrifidus leaves essential oil cytotoxic activity was significant against human lung cell line A-549 (2.37 μg), human colon cell line HCT-116 (2.39 μg), human hepato-cellular cell line HepG-2 (2.55 μg) and intestinal epithelium carcinoma Caco-2 (5.80 μg) cell lines, respectively. The oil cytotoxicity exceeded 1,8-cineole except for intestinal epithelium carcinoma Caco-2. Additionally, the antimicrobial activity was evaluated using agar disc diffusion and minimum inhibitory concentration where the oil exhibited the best activity against Penicillium italicum and Geotrichum candidum with minimum inhibitory concentration=0.49 and 0.98 μg, respectively. Structure elucidation of the pure compounds isolated from Calothamnus quadrifidus leaves was done according to their chromatographic behavior, chemical and spectroscopic data. These compounds were isolated for the first time from Calothamnus quadrifidus leaves and identified as gallic acid, gallic acid methyl ester, 7,4`-dimethoxy-apigenin, 6,7-dimethoxy-apigenin, (Bis[3(2-methyl propan-1- one)-2, 6 dihydroxy-4- methoxy phenyl] methane, quercetin 3-O-beta-D-arbinopyranoside and quercetin 3-O-beta-D-4C1-xylopyranoside. In addition to the molecular docking of novel compound 3 was done in peroxiredoxins and the binding mode was found. Conformation 2 was found to be the most stable and may be responsible for the antioxidant effect as it showed the best free binding energy and affinity when compared to conformations.

Screening of the chemoprotective effect of 13 compounds and their mixtures with sodium 2-mercaptoethanesulfonate against 2-chloroethyl ethyl sulfide.

2-chloroethyl ethyl sulfide (CEES) is a vesicant agent, commonly referred to half mustard due to its ability to form monofunctional adducts with DNA. In this study, we evaluated the chemoprotective potential of 13 compounds and their mixtures with sodium 2-mercaptoethanesulfonate (MESNA) against CEES-induced geno- and cytotoxicity in human lung cell line A-549. MESNA, L-glutathione (GSH), thiourea, sodium thiosulfate, hexamethylenetetramine, 4-acetamidophenol, asoxime dichloride (HI-6), N-acetyl-L-cysteine (NAC), sodium pyruvate, myo-inositol, 3-aminobenzamide (3-AB), nicotinamide, and Nω-nitro-L-arginine methyl ester hydrochloride and combinations of these compounds with MESNA were applied 30 min before CEES. DNA alkylation was measured using modified comet assay 1 and 24 h after the exposure. Cell viability was determined using MTT assay at 24 and 72 h. The mono-therapeutical approach identified MESNA and GSH to provide significant chemoprotection. NAC and 3-AB supported DNA damage repair, while cell viability remained unaffected. Mixtures of GSH or NAC with MESNA showed protective synergism against DNA damage. Other compounds or their combinations with MESNA failed due to the potentiation of CEES-induced cytotoxicity. The chemoprotection against CEES remains limited; however, the combination of substances can provide protective synergy and may represent a promising strategy in the treatment of accidental exposure to monoalkylating agents.

Anti-inflammatory effect of Chunkoongkeigi-tang on IL-1β-induced inflamed A549 by the inhibition of COX-2 expression

Inflammatory lung disease can arise due to the immoderate expression of pro-inflammatory genes and the disharmony of complex cytokines. Chunkoongkeigi-tang (CKT) has been prescribed to patients with asthmatic symptoms in herbal medicine clinics. However, the effects of CKT on the inflamed human lung cell line A549 have yet to be revealed. Thus we investigate whether CKT can suppress inflammatory response on interleukin 1β (IL-1β)-induced inflamed A549. In an effort to understand the inhibitory activity of CKT on inflamed A549, we use IL-1β to induce inflammatory gene COX-2 expressions on A549. Inhibition of COX-2 expression and inflammatory cytokine secretion through blocking mitogen activated protein kinase (MAPK) pathway and transcriptional nuclear factor kappa B (NF-κB) activity was confimed by western blot and real-time PCR. Compounds in CKT were identified by liquid chromatography-mass spectrometry (LC-MS). CKT inhibited increased COX-2 in an IL-1β-induced inflammatory state of A549. We confirmed that CKT regulated the mitogen activated protein kinase (MAPK) pathway and transcriptional nuclear factor kappa B (NF-κB). Additionally, a suppressive effect of CKT on the mRNA expression levels of cytokine-related asthma ((IL-5, IL-6, IL-17 and TNF-α) was noted in IL-1β-induced inflamed A549. According to LC-MS analysis, we identified several components in CKT which can be expected to be the active compounds of the anti-inflammatory effect of CKT. We verify the anti-inflammatory effect of CKT via the reduction of the IL-1β-induced increased COX-2 expression through the MAPK and NF-κB pathways, suggesting useful anti-asthmatic potential by inhibiting inflammatory cytokines, especially in patients with asthma.

Differential pulmonary in vitro toxicity of two small-sized polyvinylpyrrolidone-coated silver nanoparticles

ABSTRACTSilver nanoparticles (AgNP), with their important properties, are being used in a range of sectors from industry to medicine, leading to increased human exposure. Hence, their toxicity potential needs to be comprehensively evaluated. It was postulated that within small-sized (≤20 nm) polyvinylpyrrolidone-coated silver nanoparticles (PVP-AgNP), minor size differences may significantly induce different toxicity profiles and involve varying cellular pathways. Therefore, the aim of this study was to examine the influence of differing size AgNP with 10 nm (AgNP10) and 20 nm (AgNP20) (up to 100 µg/ml), as well as to ionic silver as AgNO3 for 24 and 48 h, using the human lung cell line A549. The effects on cell viability, proliferation, apoptosis, DNA damage and cell cycle dynamics were assessed. Results for both time periods showed that for low concentrations (<5 µg/ml), AgNP20 were more cytotoxic than AgNP10, however, at higher doses, AgNP10 exhibited higher toxicity. For concentrations >50 µg/ml, AgNP10 induced severe DNA damage (comet class 3–4), cell cycle arrest at G2 phase and late-stage apoptosis, while AgNP20 induced cell cycle arrest at S phase and an increase in the percentage sub-G1, which did not recover after 48 h, and late-stage apoptosis/necrosis. In longer-term exposures, the greater impairment in colony formation due to AgNP exposure than to silver ion supports that nanotoxicity is not exclusively due to the released ion. Data suggest that toxicity mediated by small AgNP (≤20 nm) in lung cells is not only dependent on the level of particle internalization, but also on AgNP size and concentration, which may involve varying pathways as targets.

Dust at Various Workplaces-Microbiological and Toxicological Threats.

The aim of the present study was to evaluate the relation between the chemical (analysis of elements and pH) and microbiological composition (culture and metagenomics analysis) of the dust at various workplaces (cement plant, composting plant, poultry farm, and cultivated area) and the cytotoxicity effect on the human adenocarcinoma lung epithelial adherent cell line A-549 (MTT assay test). Analysis of the Particulate Matter (PM) fraction showed that the dust concentration in cultivated areas exceeded the OELs. For the remaining workplaces examined, the dust concentration was lower than OELs limits. The number of microorganisms in the dust samples was 3.8 × 102–1.6 × 108 CFU/g bacteria and 1.5 × 102–6.5 × 106 CFU/g fungi. The highest number of microorganisms was noted for dust from cultivated areas (total number of bacteria, actinomycetes, P. fluorescens) and composting plants (xerophilic fungi and staphylococci), while the least number of microorganisms was observed for dust from cement plants. Many types of potentially pathogenic microorganisms have been identified, including bacteria, such as Bacillus, Actinomyces, Corynebacterium, Prevotella, Clostridium, and Rickettsia, and fungi, such as Alternaria, Cladosporium, Penicillium, and Aspergillus. The most cytotoxic to the human lung cell line A-549 was dust from cultivated areas (IC50 = 3.8 mg/mL after 72 h). The cytotoxicity of the tested dust samples depends on the PM concentration, the number of microorganisms, including potentially pathogenic genera, and the exposure time.

Antiproliferative effects on human lung cell lines A549 activity of cadmium selenide nanoparticles extracted from cytotoxic effects: Investigation of bio-electronic application

Cadmium selenide (CdSe) nanoparticles make necessary to acquire more information against the cytotoxic effects on human lung epithelial cells A549 potential adverse to health effects. More biological studies highlighted their cytotoxic potential like pulmonary or respiratory diseases were focused on toxicity nanoparticles mechanisms are involved. The aim of our research, is the comparison of cytotoxicity effect between cells-particle interactions, viability test, membrane integrity and oxidative stress were investigated. XRD showed a strong peak associated with (111) plane of hexagonal CdSe suggesting formation of highly orientated nanoparticles. The longitudinal optical phonon shifted slightly due to strain whereas strong low-energy shoulder shift can be explained within a model for surface optical phonons. Photocatalytic activity of CdSe nanoparticles were investigated by exploiting photocatalytic degradation of Rhodamine B (RhB). The typical UV–vis absorption spectra of RhB solution at different time intervals it can be clearly seen that the relative intensity of the absorption peak corresponding to RhB, with the catalyst for different concentration time intervals (0mM, 2mM, 5mM & 10mM) of the prepared CdSe nanoparticles. After completion of 5mM % the dye was completely degraded and the absorption spectra act as a photocatalyst. CdSe nanoparticles exhibits antibacterial activity over a broad range of bacterial species and in particular against P. vulgaris where it out competes four other commonly used S. aureus, E.coli, P. vulgaris and E. hermannii, well as testing four different appropriate concentration from the results showed a significant gain in viable cell numbers of all four bacteria species, with 5mM and 10mM being the most effective and 2mM being the worst, where it provided only a slight improvement from the control in detail.

Combined toxic effect of airborne heavy metals on human lung cell line A549.

Many studies have demonstrated that heavy metals existing as a mixture in the atmospheric environment cause adverse effects on human health and are important key factors of cytotoxicity; however, little investigation has been conducted on a toxicological study of a metal mixture from atmospheric fine particulate matter. The objective of this study was to predict the combined effects of heavy metals in aerosol by using in vitro human cells and obtain a suitable mixture toxicity model. Arsenic, nickel, and lead were selected for mixtures exposed to A549 human lung cancer cells. Cell proliferation (WST-1), glutathione (GSH), and interleukin (IL)-8 inhibition were observed and applied to the prediction models of mixture toxicity, concentration addition (CA) and independent action (IA). The total mixture concentrations were set by an IC10-fixed ratio of individual toxicity to be more realistic for mortality and enzyme inhibition tests. The results showed that the IA model was statistically closer to the observed results than the CA model in mortality, indicating dissimilar modes of action. For the GSH inhibition, the results predicted by the IA and CA models were highly overestimated relative to mortality. Meanwhile, the IL-8 results were stable with no significant change in immune reaction related to inflammation. In conclusion, the IA model is a rapid prediction model in heavy metals mixtures; mortality, as a total outcome of cell response, is a good tool for demonstrating the combined toxicity rather than other biochemical responses.

G45R on nonstructural protein 1 of influenza A virus contributes to virulence by increasing the expression of proinflammatory cytokines in mice.

Nonstructural protein 1 (NS1) is a multifunctional protein that is a viral replication enhancer and virulence factor. In this study, we investigated the effect of the amino acid substitution G45R on the NS1 of A/Puerto Rico/8/1934 (H1N1) (G45R/NS1) on viral virulence and host gene expression in a mouse model and the human lung cell line A549. The G45R/NS1 virus had increased virulence by inducing an earlier and robust proinflammatory cytokine response in mice. Mice infected with the G45R/NS1 virus lost more body weight and had lower survival rates than mice infected with the wild type (WT/NS1) virus. Replication of the G45R/NS1 virus was higher than that of the WT/NS1 virus in vitro, but the replication of both viruses was similar in mouse lungs. In A549 cells, the majority of G45R/NS1 protein was localized in the cytoplasm whereas the majority of WT/NS1 protein was localized in the nucleus. Microarray analysis revealed that A549 cells infected with the G45R/NS1 virus had higher expression of genes encoding proteins associated with the innate immune response and cytokine activity than cells infected with the WT/NS1 virus. These data agree with cytokine production observed in mouse lungs. Our findings suggest that G45R on NS1 protein contributes to viral virulence by increasing the expression of inflammatory cytokines early in infection.

Surface passivity largely governs the bioaccessibility of nickel-based powder particles at human exposure conditions

The European chemical framework REACH requires that hazards and risks posed by chemicals, including alloys and metals, are identified and proven safe for humans and the environment. Therefore, differences in bioaccessibility in terms of released metals in synthetic biological fluids (different pH (1.5–7.4) and composition) that are relevant for different human exposure routes (inhalation, ingestion, and dermal contact) have been assessed for powder particles of an alloy containing high levels of nickel (Inconel 718, 57 wt% nickel). This powder is compared with the bioaccessibility of two nickel-containing stainless steel powders (AISI 316L, 10–12% nickel) and with powders representing their main pure alloy constituents: two nickel metal powders (100% nickel), two iron metal powders and two chromium metal powders. X-ray photoelectron spectroscopy, microscopy, light scattering, and nitrogen absorption were employed for the particle and surface oxide characterization. Atomic absorption spectroscopy was used to quantify released amounts of metals in solution. Cytotoxicity (Alamar blue assay) and DNA damage (comet assay) of the Inconel powder were assessed following exposure of the human lung cell line A549, as well as its ability to generate reactive oxygen species (DCFH-DA assay). Despite its high nickel content, the Inconel alloy powder did not release any significant amounts of metals and did not induce any toxic response. It is concluded, that this is related to the high surface passivity of the Inconel powder governed by its chromium-rich surface oxide. Read-across from the pure metal constituents is hence not recommended either for this or any other passive alloy.

Role of DNA Methylation in Cell Cycle Arrest Induced by Cr (VI) in Two Cell Lines

Hexavalent chromium [Cr(IV)], a well-known industrial waste product and an environmental pollutant, is recognized as a human carcinogen. But its mechanisms of carcinogenicity remain unclear, and recent studies suggest that DNA methylation may play an important role in the carcinogenesis of Cr(IV). The aim of our study was to investigate the effects of Cr(IV) on cell cycle progress, global DNA methylation, and DNA methylation of p16 gene. A human B lymphoblastoid cell line and a human lung cell line A549 were exposed to 5–15 µM potassium dichromate or 1.25–5 µg/cm2 lead chromate for 2–24 hours. Cell cycle was arrested at G1 phase by both compounds in 24 hours exposure group, but global hypomethylation occurred earlier than cell cycle arrest, and the hypomethylation status maintained for more than 20 hours. The mRNA expression of p16 was significantly up-regulated by Cr(IV), especially by potassium dichromate, and the mRNA expression of cyclin-dependent kinases (CDK4 and CDK6) was significantly down-regulated. But protein expression analysis showed very little change of p16 gene. Both qualitative and quantitative results showed that DNA methylation status of p16 remained unchanged. Collectively, our data suggested that global hypomethylation was possibly responsible for Cr(IV) - induced G1 phase arrest,but DNA methylation might not be related to up-regulation of p16 gene by Cr(IV).

Different mechanisms for metal-induced adaptation to cadmium in the human lung cell lines A549 and H441

Sensitivity to Cd and Zn as well as the capacity to develop tolerance were characterized in human lung cells A549 and H441. In the A549 cells, a 2-fold lower LC(50) was obtained for Cd compared to Zn, whereas H441 cells were similarly sensitive to both metals. H441 cells were twice as resistant to Cd as the A549 cells. Higher HSP70, but not metallothionein (MT) or glutathione (GSH) levels, could contribute to this better resistance. A 1.5- and 2-fold increase in the LC(50) for Cd was obtained in the A549 cells pre-exposed to non-cytotoxic concentrations of Cd (20 μM) or Zn (40 μM) for 24 h. On the other hand, only Zn increased H441 cells' resistance to Cd. Maximum Zn- and Cd-induced tolerances were reached as early as 3 and 12 h, respectively. Increases in MT-IIa and HSP70 messenger RNA levels were higher in A549 cells, but cycloheximide eliminated the induction of tolerance only in the H441 cells. Protein synthesis is a prerequisite for metal-induced tolerance to Cd in the H441 cells but not the A549 cells. Results obtained with L-buthionine sulfoximine revealed that GSH synthesis is not responsible for the acquired tolerance in both cell lines. However, GSH plays a critical role against Cd toxicity, and pro-oxidant conditions sensitized cells to Cd with different impacts on the metal-induced mechanisms of acquired tolerance. GSH and catalase both provide antioxidative protection, but only the stress related to low GSH content, not that resulting from catalase inhibition, may be alleviated with Zn.

Intracellular Uptake and Toxicity of Ag and CuO Nanoparticles: A Comparison Between Nanoparticles and their Corresponding Metal Ions

An increased understanding of nanoparticle toxicity and its impact on human health is essential to enable a safe use of nanoparticles in our society. The aim of this study is to investigate the role of a Trojan horse type mechanism for the toxicity of Ag-nano and CuO-nano particles and their corresponding metal ionic species (using CuCl2 and AgNO3 ), i.e., the importance of the solid particle to mediate cellular uptake and subsequent release of toxic species inside the cell. The human lung cell lines A549 and BEAS-2B are used and cell death/membrane integrity and DNA damage are investigated by means of trypan blue staining and the comet assay, respectively. Chemical analysis of the cellular dose of copper and silver is performed using atomic absorption spectroscopy. Furthermore, transmission electron microscopy, laser scanning confocal microscopy, and confocal Raman microscopy are employed to study cellular uptake and particle-cell interactions. The results confirm a high uptake of CuO-nano and Ag-nano compared to no, or low, uptake of the soluble salts. CuO-nano induces both cell death and DNA damage whereas CuCl2 induces no toxicity. The opposite is observed for silver, where Ag-nano does not cause any toxicity, whereas AgNO3 induces a high level of cell death. CuO-nano toxicity is predominantly mediated by intracellular uptake and subsequent release of copper ions, whereas no toxicity is observed for Ag-nano due to low release of silver ions within short time periods.

Functionalised gold nanoparticles for selective induction of in vitro apoptosis among human cancer cell lines

The interaction of citrate- and polyethylene imine (PEI)-functionalised gold nanoparticles (GNP) with cancer cell lines with respect to the cellular response was studied. It was found that GNP/citrate nanoparticles were able to induce apoptosis in human carcinoma lung cell lines A549, but GNP/PEI did not show any reduction in the viability of the cells in human breast cancer cell line MCF-7 and A549 cell lines. FACS data confirmed that the number of apoptotic cells increased with increase in the concentration of GNP/citrate nanoparticles. Decline in cellular expansion and changes in the nuclear morphology were noted after the treatment of GNP/citrate nanoparticles on A549 cell lines, which itself is a direct response for stress induction. The induction of cellular apoptosis was further confirmed by DNA fragmentation assay. These data confirm the potential of GNP/citrate nanoparticle to evoke cell-specific death response in the A549 cell lines.

Effect of TiO2 nanoparticle surface functionalization on protein adsorption, cellular uptake and cytotoxicity: the attachment of PEG comb polymers using catalytic chain transfer and thiol–ene chemistry

A successful modification of titanium dioxide (TiO2) nanoparticles surfaces by a grafting-to polymer technique combining catalytic chain transfer and thiol–ene click chemistry is reported. Vinylic end functional polymers were first prepared by catalytic chain transfer polymerization (CCTP) using oligo(ethylene glycol) methacrylate as a monomer. The presence of vinylic end groups was then exploited to attach the polymers to thiol functionalized TiO2 nanoparticles via thiol–ene Michael nucleophilic reactions. X-ray photoelectron spectroscopy (XPS), attenuated total reflectance-infrared (ATR-IR), dynamic light scattering (DLS), and thermogravimetric analyses (TGA) were used to verify the successful modification of the TiO2 surface. The modified TiO2 nanoparticles were stable in cell culture media and formed smaller aggregates when compared to non-surface modified nanoparticles. Cellular toxicity of the hybrid TiO2–polymer particles towards human lung cell lines A549 and H1299 in vitro was evaluated. Results from one-dimensional gel electrophoresis show the presence of polymer layers around the particles affects the adsorption of protein onto the TiO2 surface. The reduction in particle aggregate size and changes to the particle surface chemistry, following polymer grafting, was found to reduce cellular uptake and diminish cytotoxicity for both human lung cell lines tested.

Fe- and Zn-induced inhibition of Cd uptake in human lung cell lines: Speciation studies with H441 and A549 cells

Cadmium transport was studied in human lung cell lines A549 and H441 as a function of inorganic metal speciation. A 2-fold higher equilibrium accumulation was obtained in the A549 cells, but a specific system of transport of high affinity and low capacity was characterized in both these cells. Exposure conditions optimizing [Cd2+] increased Cd uptake, but CdCln2-n species are also taken up. Studies on Cd-Ca reciprocal inhibition do not support a role for Ca pathways in Cd uptake. Nramp2 and Zip8 mRNAs were detected in both cell lines. Fe inhibited Cd uptake in the nitrate medium with an apparent at pH 5.5. NRAMP2 may contribute to Cd uptake but a major role is precluded since the overall process of accumulation was inhibited by acidic conditions. Zn and Mn inhibited Cd uptake with the following observations: (1) Similar apparent Ki values for Zn-induced inhibition in the chloride and nitrate media. (2) Whatever the medium, a higher Mn-induced inhibition at pH 5.5 compared to pH 7.4, with much lower Ki values under acidic conditions. (3) Elimination of the stimulatory effect of HCO3- by Zn but not by Mn. Zn may inhibit a ZIP8-mediated Cd uptake, whereas the Mn-sensitive component of uptake would be related to other transport processes.

Abstract 4817: Altered DNA methylation in human lung cell line A549 after treatment with a standard mixture of particulate matter

Abstract BACKGROUND: Numerous health studies have shown acute and chronic particulate air pollution (PM, particulate matter) exposures to be associated with lung cancer, as well as with lung growth impairment, chronic obstructive pulmonary disease (COPD), and asthma. Toxicology studies have shown that PM is only a weak mutagen. Whether PM induces DNA methylation alterations, a primary mechanism in cell transformation, has never been studied in lung epithelial cells. In our study, we found a significative modification of P16 DNA methylation after PM treatement. AIM: To investigate PM on 8 cancer and inflammatory genes (TLR-4, CD14, IFNγ, TNFα, RASSF1A, APC, p53, p16) in A549 human alveolar basal epithelial cells. METHODS: A549 cells were treated with a complex standard mixture of particulate matter (NIST-National Institute of Standards and Technology- Standard Reference Material [SRM] 1648a). SRM 1648a treatments were performed with increasing doses of total mixture (15, 31, 62, 125, and 250 μg/ml) dissolved in colture medium. We used untreated cells as a negative control. Only concentrations that did not affect cell viability were considered. The cells were harvested after 2, 6, 12, 24, and 48 hours of continuous exposure, washed with phosphate buffered saline, and stored as cell pellet at -80°C until DNA extraction. DNA methylation was measured by means of bisulfite-Pyrosequencing. All the experiments were conducted in triplicate to confirm repeatability of our results. RESULTS: Using a t-test assuming that the two distributions have the same variance, we found an increase in p16 methylation for a dose of 15 μg/ml (p16 mean(untreated-treated)=0,83; p-value=0,05); an hypomethylation for a dose of 62 μg/ml (p16 mean(untreated-treated)=−0,77; p-value=0,004) as well as for a dose of 250 μg/ml (p16 mean(untreated-treated)=−0,78; p-value=0,01). We observed also an increase in APC methylation for a dose of 250 μg/ml (APC mean(untreated-treated)=2,90; p-value=0,02). CONCLUSION: This finding indicates that different levels of exposure to PM can induce changes in the DNA methylation levels in vitro. Our hypothesis is that exposure to PM can cause changes in the gene-specific DNA methylation levels thus enhancing cancerogenesis. Additional studies are required to better define the epigenetic mechanisms by which PM and similar environmental agents induce changes in DNA methylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4817. doi:10.1158/1538-7445.AM2011-4817

Biological impacts of TiO2 on human lung cell lines A549 and H1299: particle size distribution effects

Increasing use of titanium dioxide (TiO2) nanoparticles in many commercial applications has led to emerging concerns regarding the safety and environmental impact of these materials. In this study, we have investigated the biological impact of nano-TiO2 (with particle primary size of 20 nm Aeroxide P25) on human lung cell lines in vitro and also the effect of particle size distribution on the particle uptake and apparent toxicity. The biological impact of nano-TiO2 is shown to be influenced by the concentration and particle size distribution of the TiO2 and the impact was shown to differ between the two cell lines (A549 and H1299) investigated herein. A549 cell line was shown to be relatively resistant to the total amount of TiO2 particles uptaken, as measured by cell viability and metabolic assays, while H1299 had a much higher capacity to ingest TiO2 particles and aggregates, with consequent evidence of impact at concentrations as low as 30–150 μg/mL TiO2. Evidence gathered from this study suggests that both viability and metabolic assays (measuring metabolic and mitochondrial activities and also cellular ATP level) should be carried out collectively to gain a true assessment of the impact of exposure to TiO2 particles.