Engineered nanomaterials cause cytotoxicity and activation on mouse antigen presenting cells.

Nanomaterials improve everyday products but their safety for human health is poorly known. In this study we explored immunological effects of five different nanomaterials on antigen presenting cells (APC) in vitro. Nanomaterials studied were rutile titanium dioxide (TiO2), amorphous silica-coated rutile titanium dioxide (TiO2-silica), zinc oxide (ZnO), single-walled carbon nanotubes (SWCNT) and multi-walled carbon nanotubes (MWCNT). APCs included mouse macrophages (RAW 264.7 cell line) and murine bone marrow-derived dendritic cells (bmDC). All studied particles were cytotoxic to bmDCs, and ZnO, TiO2 and TiO2-silica-induced dose-dependently cell death also in macrophages. ZnO had the most drastic immunological effects leading to high expression of proinflammatory cytokine, IL-1beta, and enhanced production of neutrophil chemoattractant CXCL-9 on both cell types. TiO2 and TiO2-silica stimulated the expression of IL-6, MIP-1alpha and TNF-alpha in macrophages, and increased their maturation, antigen presentation and co-stimulation activity. In contrast, SWCNT or MWCNT did not seem to have any significant immunological effects on the cell types studied suggesting that APCs might not be the target cells for carbon nanotubes. Due to diverse effects on different nanomaterials on immune cells we suggest that each new nanomaterial should be extensively studied in vitro and in vivo for risk assessment before their use in final products.

Preparation of nanoparticle dispersions for in-vitro toxicity testing.

Studies on potential toxicity of engineered nanoparticle (ENP) in biological systems require a proper and accurate particle characterization to ensure the reproducibility of the results and to understand biological effects of ENP. A full characterization of ENP should include various measurements such as particle size and size distribution, shape and morphology, crystallinity, composition, surface chemistry, and surface area of ENP. It is also important to characterize the state of ENP dispersions. In this study, four different ENPs, rutile and anatase titanium dioxides and short single- and multi-walled carbon nanotubes, were characterized in two dispersion media: bronchial epithelial growth medium, used for bronchial epithelial BEAS cells, and RPMI-1640 culture media with 10% of fetal calf serum (FCS) for human mesothelial (MeT-5A) cells. The purpose of this study was to determine the characteristics of ENPs and their dispersions as well as to compare dispersion additives suitable for toxicity tests and thus establish an appropriate way to prepare dispersions that performs well with the selected ENP. Dispersion additives studied in the media were bovine serum albumin (BSA) as a protein resource, dipalmitoyl phosphatidylcholine (DPPC) as a model lung surfactant, and combination of BSA and DPPC. Dispersions were characterized using optical microscopy and transmission electron microscopy. Our results showed that protein addition, BSA or FCS, in cell culture media generated small agglomerates of primary particles with narrow size variations and improved the stability of the dispersions and thus also the relevance of the in-vitro genotoxicity tests to be done.

Effects of fumonisin B(1) on the expression of cytokines and chemokines in human dendritic cells.

Fumonisin B(1) (FB(1)) is a mycotoxin produced by the fungus Fusarium verticillioides, which commonly infects corn and other crops across the world. Exposure to FB(1) is known to have toxic and carcinogenic effects in animals, and to express toxicity in cells. In this study, we investigated mechanisms whereby FB(1) may induce immunotoxic effects in human dendritic cells (DC) differentiated from human peripheral blood mononuclear cells. mRNA and protein levels of a number of cytokines and chemokines were analyzed in DC, after exposure to 100 microM FB(1), 10 ng/ml LPS, or a combination of 100 microM FB(1) and 10 ng/ml LPS for 6h or 24h. Exposure to FB(1) resulted in an increase in the expression of IFNgamma and CXCL9. Moreover, FB(1) inhibited the LPS-induced expression of IL-6, IL-1beta, CCL3 and CCL5. The other cytokines studied (TNFalpha, IL-12, IL-18 and IL-23) were not affected by FB(1) in DC. The results of this study indicate that FB(1) has an impact on the expression of cytokines and chemokines in human DC, and in addition to its other toxic effects, FB(1) may also be potentially immunotoxic to humans.

A review of the toxic effects and mechanisms of action of fumonisin B1.

Fumonisin B(1) (FB(1)) is a mycotoxin produced by the fungus Fusarium verticillioides, which commonly infects corn and other agricultural products. Fusarium species can also be found in moisture-damaged buildings, and, therefore, exposure of humans to Fusarium mycotoxins including FB(1) may take place. FB(1) bears a clear structural similarity to the cellular sphingolipids, and this similarity has been shown to disturb the metabolism of sphingolipids by inhibiting the enzyme ceramide synthase leading to accumulation of sphinganine in cells and tissues. FB(1) is neurotoxic, hepatotoxic, and nephrotoxic in animals, and it has been classified as a possible carcinogen to humans. The cellular mechanisms behind FB(1)-induced toxicity include the induction of oxidative stress, apoptosis, and cytotoxicity, as well as alterations in cytokine expression. The effects of FB(1) on different parameters vary markedly depending on what types of cells are studied or what species they originate from. These aspects are important to consider when evaluating the toxic potential of FB(1).

In mouse alpha -methylacyl-CoA racemase, the same gene product is simultaneously located in mitochondria and peroxisomes.

alpha-Methylacyl-CoA racemase, an enzyme of the bile acid biosynthesis and branched chain fatty acid degradation pathway, was studied at the protein, cDNA, and genomic levels in mouse liver. Immunoelectron microscopy and subcellular fractionation located racemase to mitochondria and peroxisomes. The enzymes were purified from both organelles with immunoaffinity chromatography. The isolated proteins were of the same size, with identical N-terminal amino acid sequences, and the existence of additional proteins with alpha-methylacyl-CoA racemase activity was excluded. A racemase gene of about 15 kilobases was isolated. Southern blot analysis and chromosomal localization showed that only one racemase gene is present, on chromosome 15, region 15B1. The putative initial ATG in the racemase gene was preceded by a functional promotor as shown with the luciferase reporter gene assay. The corresponding cDNAs were isolated from rat and mouse liver. The recombinant rat protein was overexpressed in active form in Pichia pastoris. The presented data suggest that the polypeptide encoded by the racemase gene can alternatively be targeted to peroxisomes or mitochondria without modifications. It is concluded that the noncleavable N-terminal sequence of the polypeptide acts as a weak mitochondrial and that the C-terminal sequence acts as a peroxisomal targeting signal.

Nitric oxide and proinflammatory cytokines in nasal lavage fluid associated with symptoms and exposure to moldy building microbes.

Epidemiological data indicate that living or working in a moldy building is associated with increased risk of respiratory symptoms and disease related to inflammatory reactions, but biochemical evidence linking cause and effect is still scarce. The staff working in a mold-contaminated school, and a reference group without such exposure, were studied. Nasal lavage was performed and health data were collected with a questionnaire at the end of the spring term, after a 2.5-mo summer vacation, and at the end of the fall term. Here we show that concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO) in nasal lavage fluid were significantly higher in the exposed than in the control subjects at the end of the first exposure period. These inflammatory mediators decreased to reference group concentrations during the period when there was no exposure and the production of NO and IL-6 increased again during the reexposure in the fall term. Reports of cough, phlegm, rhinitis, eye irritation, and fatigue paralleled the changes in the measured inflammatory markers. These results point to an association between inflammatory markers in the nasal lavage fluid, the high prevalence of respiratory symptoms among the occupants, and chronic exposure to molds in the indoor environment.

Measurement of prostate-specific antigen in detection of benign or malignant breast disease in women.

Using a highly sensitive chemiluminescent enzyme immunoassay, we have evaluated the measurement of serum prostate-specific antigen (PSA) as a potential diagnostic test for differentiation between women with breast cancer and those with benign breast disease. In a controlled study consisting of 284 women with well-documented patient files and matched for age and long-term place of residence, serum samples collected from 90 women with histologically confirmed breast cancer, 94 women with benign breast disease and 100 controls were analysed. Serum total PSA levels in benign breast disease and cancer patients are not statistically different from those of healthy controls. Total PSA levels decrease with age in normal controls and breast cancer patients but not in those with benign breast disease. The total PSA concentration decreases after menopause in healthy women, though not in patients with breast cancer or benign breast disease. Total PSA bore no relation to the histological type or grade of the tumour or the disease stage of the breast cancer patients. In benign breast disease, all mastopathy patients had normal total PSA, whereas elevation of the values was observed in 7% of fibroadenoma patients. Our results show that serum total PSA cannot be used to distinguish between healthy women and/or women with breast cancer or benign breast disease.

Streptomyces anulatus from indoor air of moldy houses induce NO and IL-6 production in a human alveolar epithelial cell-line.

Moisture associated microbial growth in buildings may cause respiratory symptoms such as pulmonary inflammation. We studied the effects of spores of Streptomyces anulatus, commonly found in moldy buildings, on the production of nitric oxide (NO), interleukin-6 (IL-6), interleukin-5 (IL-5), interleukin-4 (IL-4), and tumour necrosis factor alpha (TNFα), as well as cell viability in human alveolar II type epithelial cell line (A549). Cells were exposed in vitro to S. anulatus spores with and without interferon-γ (IFNγ) in vitro. Lipopolysaccharide (LPS) was used as a reference substance. S. anulatus alone, and in combination with IFNγ induced NO and IL-6 production and decreased cell viability whereas IL-4, IL-5 or TNFα production were not affected. IFNγ alone had a weaker but otherwise similar effect as S. anulatus on NO and IL-6 production and it potentiated the effects of S. anulatus. LPS did not induce NO or cytokine production, or affect cell viability in A549 cells. These data indicate that spores of S. anulatus induce the excretion of inflammatory mediators in respiratory epithelial cells, which may partly explain the adverse respiratory health effects experienced by individuals exposed to the indoor air of moldy houses.

Glutamate-stimulated ROS production in neuronal cultures: interactions with lead and the cholinergic system.

Oxidative stress may be an important factor in several pathological brain conditions. A contributing factor in many such conditions is excessive glutamate release, and subsequent glutamatergic neuronal stimulation, that causes increased production of reactive oxygen species (ROS), oxidative stress, excitotoxicity and neuronal damage. Glutamate release is also associated with illnesses such as Alzheimer's disease, stroke, and brain injury. Glutamate may interact with an environmental toxin, lead, and this interaction may result in neuronal damage. Glutamate-induced ROS production is greatly amplified by lead in cultured neuronal cells. Alterations in protein kinase C (PKC) activity seem to be important both for glutamate-induced ROS production, and for the amplification of glutamate-induced ROS production by lead. It is possible that the neurotoxic effects of lead are amplified through glutamate-induced neuronal excitation. Cholinergic stimulation can also trigger ROS production in neuronal cells. PKC seems to play a key-role also in cholinergic-induced ROS production superoxide anion being the primary reactive oxygen species. There seems to be a close relationship between the responses of cholinergic muscarinic and glutamatergic receptors because glutamate receptor antagonists inhibit cholinergic-induced activation of human neuroblastoma cells. Glutamatergic neuronal stimulation may be a common final pathway in several brain conditions in which oxidative stress and ensuing excitotoxicity plays a role.

Dissolution of short and long rockwool and glasswool fibers by macrophages in flowthrough cell culture.

Dissolution of MMVF (man-made vitreous fibers) by macrophages has previously been studied utilizing cell cultures in wells. A new, more dynamic method has been developed to explore the effects of macrophages on MMVF dissolution. In this method, the culture medium flows through a membrane on which the macrophages and fibers are placed. The dissolution of short and long rockwool and glasswool fibers was investigated in the present study by macrophages by assessing the dissolution of Si (silicon), Fe (iron), and Al (aluminium) from the fibers. Dissolution of these elements usually increased as a function of time. Generally, the dissolution of elements from the fibers in the flowthrough culture exceeded that observed with the culture in wells system. The dissolution of glasswool fibers was greater in medium than in cell culture, whereas the opposite was true for rockwool fibers. Dissolution of Si was greater from glasswool than from rockwool fibers, while the opposite was true for Fe and Al. Macrophages that had phagocytized fibers in flowthrough culture contained Si, and there were also precipitations with Si in the samples. The fibers in the flowthrough culture also exhibited surface changes such as breakings, pittings, etching, and peeling. The short rockwool fibers tended to fracture more than short glasswool fibers, while long glasswool fibers were more extensively broken than short glasswool fibers. The results with this new, dynamic, flowthrough culture method with macrophages demonstrate that this method provides valuable information on the abilities of macrophages to dissolve MMVF leading to subsequent morphological changes of fibers.

Modification of glutamate-induced oxidative stress by lead: the role of extracellular calcium.

The role of extracellular calcium in glutamate-induced oxidative stress, and the role of glutamatergic neuronal stimulation and oxidative stress in lead neurotoxicity were explored in mouse hypothalamic GT1-7 cells. Glutamate increased the production of reactive oxygen species (ROS) whether or not extracellular calcium was present. Glutamate-induced ROS production was amplified by lead acetate (PbAc), but only in the absence of extracellular calcium. However, PbAc on its own did not increase the production of ROS. A PKC inhibitor (Ro 31-8220) and superoxide dismutase (SOD) abolished the amplification of glutamate-induced production of ROS by PbAc, but did not inhibit ROS production induced by glutamate alone. Both glutamate and PbAc decreased the levels of intracellular glutathione (GSH), and amplified each other's effect on GSH depletion. Glutamate did not decrease cell viability, whereas the cytotoxicity of PbAc was amplified by glutamate. Extracellular calcium, a PKC inhibitor, or SOD did not modify the effects of glutamate, PbAc or their combination on the levels of GSH or cell viability. These data indicate that in GT1-7 cells extracellular calcium is not essential for glutamate-induced ROS production, which is amplified by PbAc, but only without extracellular calcium. The joint cytotoxicity of glutamate and PbAc is mainly induced by PbAc, preferentially through mechanisms other than ROS production.

Cytotoxicity, production of reactive oxygen species and cytokines induced by different strains of Stachybotrys sp. from moldy buildings in RAW264.7 macrophages.

The ability of different strains of the fungus Stachybotrys, isolated from mold problem buildings, to induce cytotoxicity and production of important inflammatory mediators, i.e. nitric oxide (NO), reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in RAW264.7 macrophages were studied. Several strains of Stachybotrys sp. stimulated immediate increase in the ROS production and in 24-h exposure caused TNF-α and IL-6 release from these cells. However, none of the strains of Stachybotrys sp. was able to induce the expression of inducible nitric oxide synthase (iNOS) and subsequent production of NO in RAW264.7 cells. Moreover, there were significant differences in their ability to induce cytotoxicity in the macrophages. These results suggest that, in addition to direct cytotoxic effects of most Stachybotrys sp., some strains of Stachybotrys sp. stimulate production of inflammatory mediators, TNF-α and IL-6 which were associated with low cytotoxicity in RAW264.7 macrophages.

Species differences in NO formation by rat and hamster alveolar macrophages in vitro.

Nitric oxide (NO) is a cellular mediator and regulator of multiple biologic functions. NO released by alveolar macrophages (AM) is suggested to play a role in mediating pulmonary injury. In murine and rat macrophages, the expression of inducible NO synthase (iNOS) and the release of NO are well established. However, the existence of such a pathway in other species remains controversial. In this study, we examined NO production and iNOS expression by AM from rats and hamsters, two laboratory animal species that are characterized by their disparate pulmonary responses to various inhaled irritants/toxicants. AM were treated with lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), or tumor necrosis factor-alpha (TNF-alpha) in vitro, and nitrite, the stable oxidation product of NO, was assayed by the Griess reaction. Rat AM produced NO in a dose- and time-dependent manner upon stimulation with LPS and/or IFN-gamma, but not with TNF-alpha. Surprisingly, hamster AM did not release detectable levels of NO after the same treatment. Although iNOS expression was demonstrated in rat AM by immunocytochemical and Western blot analyses, no induction of iNOS expression could be found in hamster AM. Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we found that rat and hamster AM could be induced to express iNOS mRNA after treatment with LPS and IFN-gamma. The results presented here indicate that hamster AM, in contrast to rat AM, lack the ability to express iNOS protein and to generate NO in response to LPS, IFN-gamma, or TNF-alpha in vitro. In conclusion, our data suggest striking differences in iNOS regulation and NO production by AM from rats and hamsters, two rodent species that are commonly used in biomedical research and well-known for their disparate responses to pulmonary irritants/toxicants.

Plasminogen receptors. Turning Salmonella and Escherichia coli into proteolytic organisms.

We evaluated in vitro the hypothesis that bacterial adhesiveness to the mammalian extracellular matrix and the activation of plasminogen on bacterial plasminogen receptors promote bacterial penetration through basement membranes. We used the strain SH401 of Salmonella enterica serovar Typhimurium, which adheres to the high-mannose chains of laminin, a major glycoprotein of basement membranes, and expresses plasminogen receptors. Bacterium-bound plasmin was able to degrade laminin and extracellular matrix preparations as well as to potentiate the penetration of bacteria through a reconstituted basement membrane. The results suggest that metastatic tumour cells and bacterial pathogens use similar mechanisms to penetrate through tissue barriers.

Cholinergic-induced production of reactive oxygen species in human neuroblastoma cells.

Stimulation of human SH-SY5Y neuroblastoma cells by a muscarinic receptor agonist, carbachol (CCh; 1 mM), elevated levels of free intracellular calcium and subsequently increased the production of reactive oxygen species (ROS). Quinuclidinylbenzilate (QNB) binding increased at 1 h after CCh, but returned back to the control level at 3 h. Production of ROS increased, however, during the 3 h time period. CCh also increased the translocation of protein kinase C (PKC) to the membrane. ROS production was completely blocked by atropine and a PKC inhibitor, Ro 31-8220. These results show that increased ROS production was a result of muscarinic receptor stimulation, and that PKC had an active role in this cellular stimulation. ROS production upon cellular stimulation by CCh was completely inhibited also by superoxide dismutase, and partially by catalase, indicating that the formation of superoxide anion dominated in cholinergic-induced generation of ROS in human neuroblastoma cells. These results also show that muscarinic stimulation causes sustained ROS production in human neuroblastoma cells. The slow increase in ROS production by CCh suggest a stepwise cascade of events leading to oxidative stress with a triggering role of cholinergic muscarinic receptors in this process.

Palmitic acid anilide-induced respiratory burst in human polymorphonuclear leukocytes is inhibited by a protein kinase C inhibitor, Ro 31-8220.

Human polymorphonuclear leukocytes (PMNL) were exposed to palmitic acid anilide, an impurity in the case oils that caused the Spanish Toxic Oil Syndrome in 1981, and to the corresponding fatty acid, palmitic acid. The effects of these compounds were studied on the production of reactive oxygen metabolites (ROM) and changes in the levels of free intracellular calcium. Palmitic acid anilide induced the production of reactive oxygen metabolites in PMNL. Interestingly, the palmitic acid anilide-induced respiratory burst was completely blocked by a protein kinase C inhibitor, Ro 31-8220. Moreover, palmitic acid anilide additively amplified the production of ROM caused by a chemotactic peptide, formyl-Methionyl-Leucyl-Phenylalanine (FMLP). In contrast, palmitic acid anilide did not have any effect on the production of ROM induced by a tumor promoter, phorbol myristate acetate (PMA). Palmitic acid, in turn, did not markedly induce the production of ROM nor did it amplify the agonist-induced respiratory burst. Neither of the compounds, alone or in combination with FMLP, affected the levels of intracellular calcium in PMNL. These results indicate that the aniline moiety in palmitic acid modifies its effects on the activation of human PMNL, and the subsequent oxidative burst. The present results also suggest that palmitic acid anilide may activate PMNL through a protein kinase C-dependent mechanism.

Effect of viability of actinomycete spores on their ability to stimulate production of nitric oxide and reactive oxygen species in RAW264.7 macrophages.

Spores of actinomycetes, mesophilic gram-positive bacteria, isolated from moldy houses, induced the expression of inducible NO-synthase (iNOS) with a subsequent NO-production in RAW264.7 macrophages. No differences were detected between production of nitric oxide (NO) by alive or irradiated spores of different strains of Actinomycetes sp. or Streptomyces sp. Moreover, a significant production of reactive oxygen species (ROS) occurred in the macrophages after their stimulation both by alive and irradiation killed spores of actinomycetes. However, ROS-responses in macrophage induced by dead spores were significantly lower compared to those induced by alive spores. The cytotoxicity of the spores of different actinomycetes differed widely. The production of NO and ROS did not depend directly on the viability of the spores, suggesting an important role for cell wall components in the activation of the cells.

Induced production of nitric oxide, tumor necrosis factor, and interleukin-6 in RAW 264.7 macrophages by streptomycetes from indoor air of moldy houses.

Dampness and mold growth in buildings cause spore generation into indoor air, which is associated with respiratory tract disorders. Specific agents or cellular mechanisms of diseases have not yet been identified. In this study, airborne spores of Streptomyces sp., isolated from moldy houses, stimulated RAW264.7 macrophages, which produced tumor necrosis factor alpha and interleukin-6 and induced the expression of inducible nitric oxide synthase, with subsequent nitric oxide production. Spores of other microorganisms typically found in moldy houses did not markedly increase the production of these inflammatory mediators. The data implied a mechanism by which Streptomyces sp. may lead to respiratory tract disorders in individuals who live in moldy houses.

Streptomyces spores from mouldy houses induce nitric oxide, TNFα and IL-6 secretion from RAW264.7 macrophage cell line without causing subsequent cell death.

The current view is that only bacterial lipopolysaccharide (LPS) and gamma interferon (IFNγ) are able to alone activate macrophages to secrete nitric oxide (NO), probably a causative agent of cell death. Moreover, some cytokines and gram positive pathogens together with IFNγ induce NO-production. Surprisingly, spores of Streptomyces sp., which are mesophilic gram-positive bacteria found in mouldy houses, stimulated RAW264.7 macrophages to produce pro-inflammatory cytokines, tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6), and induced the expression of inducible NO-synthase (iNOS) with a subsequent NO-production. However, the Streptomyces spores did not kill NO-producing macrophages, as did both LPS and gram negative bacteria Pseudomonas fluorescens, strong inducers of cytokine- and NO-production. These results imply that Streptomyces sp., induced cytokine and NO-secretion, may play a role in the responses evoked by exposure to these microbes. Moreover, factors other than, or in addition to NO, are necessary for cytotoxicity in murine macrophages.