Multi-walled carbon nanotubes directly induce epithelial-mesenchymal transition in human bronchial epithelial cells via the TGF-ß-mediated Akt/GSK-3ß/SNAIL-1 signalling pathway.

BACKGROUND: Multi-walled carbon nanotubes (MWCNT) are currently under intense toxicological investigation due to concern on their potential health effects. Current in vitro and in vivo data indicate that MWCNT exposure is strongly associated with lung toxicity (inflammation, fibrosis, granuloma, cancer and airway injury) and their effects might be comparable to asbestos-induced carcinogenesis. Although fibrosis is a multi-origin disease, epithelial-mesenchymal transition (EMT) is recently recognized as an important pathway in cell transformation. It is known that MWCNT exposure induces EMT through the activation of the TGF-ß/Smad signalling pathway thus promoting pulmonary fibrosis, but the molecular mechanisms involved are not fully understood. In the present work we propose a new mechanism involving a TGF-ß-mediated signalling pathway. METHODS: Human bronchial epithelial cells were incubated with two different MWCNT samples at various concentrations for up to 96 h and several markers of EMT were investigated. Quantitative real time PCR, western blot, immunofluorescent staining and gelatin zymographies were performed to detect the marker protein alterations. ELISA was performed to evaluate TGF-ß production. Experiments with neutralizing anti-TGF-ß antibody, specific inhibitors of GSK-3ß and Akt and siRNA were carried out in order to confirm their involvement in MWCNT-induced EMT. In vivo experiments of pharyngeal aspiration in C57BL/6 mice were also performed. Data were analyzed by a one-way ANOVA with Tukey's post-hoc test. RESULTS: Fully characterized MWCNT (mean length < 5 µm) are able to induce EMT in an in vitro human model (BEAS-2B cells) after long-term incubation at sub-cytotoxic concentrations. MWCNT stimulate TGF-ß secretion, Akt activation and GSK-3ß inhibition, which induces nuclear accumulation of SNAIL-1 and its transcriptional activity, thus contributing to switch on the EMT program. Moreover, a significant increment of nuclear ß-catenin - due to E-cadherin repression and following translocation to nucleus - likely reinforces signalling for EMT promotion. In vivo results supported the occurrence of pulmonary fibrosis following MWCNT exposure. CONCLUSIONS: We demonstrate a new molecular mechanism of MWCNT-mediated EMT, which is Smad-independent and involves TGF-ß and its intracellular effectors Akt/GSK-3ß that activate the SNAIL-1 signalling pathway. This finding suggests potential novel targets in the development of therapeutic and preventive approaches.

HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast.

BACKGROUND: Invasive micropapillary carcinoma (IMPC) of the breast is a distinct and aggressive variant of luminal type B breast cancer that does not respond to neoadjuvant chemotherapy. It is characterized by small pseudopapillary clusters of cancer cells with inverted cell polarity. To investigate whether hypoxia-inducible factor-1 (HIF-1) activation may be related to the drug resistance described in this tumor, we used MCF7 cancer cells cultured as 3-D spheroids, which morphologically simulate IMPC cell clusters. METHODS: HIF-1 activation was measured by EMSA and ELISA in MCF7 3-D spheroids and MCF7 monolayers. Binding of HIF-1α to MDR-1 gene promoter and modulation of P-glycoprotein (Pgp) expression was evaluated by ChIP assay and FACS analysis, respectively. Intracellular doxorubicin retention was measured by spectrofluorimetric assay and drug cytotoxicity by annexin V-FITC measurement and caspase activity assay. RESULTS: In MCF7 3-D spheroids HIF-1 was activated and recruited to participate to the transcriptional activity of MDR-1 gene, coding for Pgp. In addition, Pgp expression on the surface of cells obtained from 3-D spheroids was increased. MCF7 3-D spheroids accumulate less doxorubicin and are less sensitive to its cytotoxic effects than MCF7 cells cultured as monolayer. Finally, HIF-1α inhibition either by incubating cells with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (a widely used HIF-1α inhibitor) or by transfecting cells with specific siRNA for HIF-1α significantly decreased the expression of Pgp on the surface of cells and increased the intracellular doxorubicin accumulation in MCF7 3-D spheroids. CONCLUSIONS: MCF7 breast cancer cells cultured as 3-D spheroids are resistant to doxorubicin and this resistance is associated with an increased Pgp expression in the plasma membrane via activation of HIF-1. The same mechanism may be suggested for IMPC drug resistance.

Modulation of doxorubicin resistance by the glucose-6-phosphate dehydrogenase activity.

How anti-neoplastic agents induce MDR (multidrug resistance) in cancer cells and the role of GSH (glutathione) in the activation of pumps such as the MRPs (MDR-associated proteins) are still open questions. In the present paper we illustrate that a doxorubicin-resistant human colon cancer cell line (HT29-DX), exhibiting decreased doxorubicin accumulation, increased intracellular GSH content, and increased MRP1 and MRP2 expression in comparison with doxorubicin-sensitive HT29 cells, shows increased activity of the PPP (pentose phosphate pathway) and of G6PD (glucose-6-phosphate dehydrogenase). We observed the onset of MDR in HT29 cells overexpressing G6PD which was accompanied by an increase in GSH. The G6PD inhibitors DHEA (dehydroepiandrosterone) and 6-AN (6-aminonicotinamide) reversed the increase of G6PD and GSH and inhibited MDR both in HT29-DX cells and in HT29 cells overexpressing G6PD. In our opinion, these results suggest that the activation of the PPP and an increased activity of G6PD are necessary to some MDR cells to keep the GSH content high, which is in turn necessary to extrude anticancer drugs out of the cell. We think that our data provide a new further mechanism for GSH increase and its effects on MDR acquisition.

Antioxidants prevent the RhoA inhibition evoked by crocidolite asbestos in human mesothelial and mesothelioma cells.

Asbestos is a naturally occurring fibrous silicate, whose inhalation is highly related to the risk of developing malignant mesothelioma (MM), and crocidolite is one of its most oncogenic types. The mechanism by which asbestos may cause MM is unclear. We have previously observed that crocidolite in human MM (HMM) cells induces NF-κB activation and stimulates the synthesis of nitric oxide by inhibiting the RhoA signaling pathway. In primary human mesothelial cells (HMCs) and HMM cells exposed to crocidolite asbestos, coincubated or not with antioxidants, we evaluated cytotoxicity and oxidative stress induction (lipid peroxidation) and the effect of asbestos on the RhoA signaling pathway (RhoA GTP binding, Rho kinase activity, RhoA prenylation, hydroxy-3-methylglutharyl-CoA reductase activity). In this paper we show that the reactive oxygen species generated by the incubation of crocidolite with primary HMCs and three HMM cell lines mediate the inhibition of 3-hydroxy-3-methylglutharyl-CoA reductase (HMGCR). The coincubation of HMCs and HMM cells with crocidolite together with antioxidants, such as Tempol, Mn-porphyrin, and the association of superoxide dismutase and catalase, prevented the cytotoxicity and lipoperoxidation caused by crocidolite alone as well as the decrease of HMGCR activity and restored the RhoA/RhoA-dependent kinase activity and the RhoA prenylation. The same effect was observed when the oxidizing agent menadione was administrated to the cells in place of crocidolite. Such a mechanism could at least partly explain the effects exerted by crocidolite fibers in mesothelial cells.

Does vitreous silica contradict the toxicity of the crystalline silica paradigm?

"Vitreous silica" is a particular form of amorphous silica, much neglected in experimental studies on silica toxicity. In spite of the incorrect term "quartz glass", often employed, this material is fully amorphous. When reduced in powdered form by grinding, the particulate appears most close to workplace quartz dust but, opposite to quartz, is not crystalline. As silicosis and lung cancer are also found among workers exposed to "quartz glass", the question arises of whether crystallinity is the prerequisite feature that makes a silica dust toxic. We compare here the behavior of comminuted quartz, vitreous silica, and monodispersed silica spheres, as it concerns surface reactivity and cellular responses involved in the accepted mechanisms of silica toxicity. Care was taken to choose samples of extreme purity, to avoid any effect due to trace contaminants. Quartz and vitreous silica, opposite to silica spheres, show irregular particles with sharp edges, stable surface radicals, and sustained release of HO(*) radicals via a Fenton-like mechanism. The evolution of the heat of adsorption of water as a function of coverage shows with quartz and vitreous silica a similar pattern of strong hydrophilic sites, nearly absent on the other silica specimen. When tested on a macrophage cell line (MH-S), vitreous silica and pure quartz, but not the monodispersed silica spheres, showed a remarkable potency in cytotoxicity, nitric oxide synthase activation and release of nitrite, and tumor necrosis factor-alpha production, suggesting a common behavior in inducing an oxidative stress. All of the above features appear to indicate that crystallinity might not be a necessary prerequisite to make a silica particle toxic.

Digoxin and ouabain induce P-glycoprotein by activating calmodulin kinase II and hypoxia-inducible factor-1alpha in human colon cancer cells.

Digoxin and ouabain are cardioactive glycosides, which inhibit the Na+/K+-ATPase pump and in this way they increase the intracellular concentration of cytosolic calcium ([Ca2+](i)). They are also strong inducers of the P-glycoprotein (Pgp), a transmembrane transporter which extrudes several drugs, including anticancer agents like doxorubicin. An increased amount of Pgp limits the absorption of drugs through epithelial cells, thus inducing resistance to chemotherapy. The mechanism by which cardioactive glycosides increase Pgp is not known and in this work we investigated whether digoxin and ouabain elicited the expression of Pgp with a calcium-driven mechanism. In human colon cancer HT29 cells both glycosides increased the [Ca2+](i) and this event was dependent on the calcium influx via the Na+/Ca2+ exchanger. The increased [Ca2+](i) enhanced the activity of the calmodulin kinase II enzyme, which in turn activated the transcription factor hypoxia-inducible factor-1alpha. This one was responsible for the increased expression of Pgp, which actively extruded doxorubicin from the cells and significantly reduced the pro-apoptotic effect of the drug. All the effects of glycosides were prevented by inhibiting the Na+/Ca2+ exchanger or the calmodulin kinase II. This work clarified the molecular mechanisms by which digoxin and oubain induce Pgp and pointed out that the administration of cardioactive glycosides may widely affect the absorption of drugs in colon epithelia. Moreover, our results suggest that the efficacy of chemotherapeutic agent substrates of Pgp may be strongly reduced in patients taking digoxin.

Formation of a vitreous phase at the surface of some commercial diatomaceous earth prevents the onset of oxidative stress effects.

To understand the effect of the commercial processing of diatomaceous earths (DEs) on their ultimate surface structure and potential toxicity, we investigated the influence of the industrial processing and the nature of the deposit. Two flux calcined specimens from different deposits, DE/1-FC and DE/2-FC, and the simply calcined sample DE/1-C, from the same deposit as DE/1-FC, were compared in both their bulk and their surface properties. X-ray diffraction (XRD) analysis in a heating chamber revealed the presence of cristobalite in all samples, more abundant on the flux calcined ones. The crystal lattice is probably imperfect, as the alpha-beta transition, visible by XRD in DE/1-FC and DE/2-FC, is not detected by differential scanning calorimetry. Progressive etching with HF solutions suggests that most of the crystalline phase is at the core and not at the outer region of the samples. The combined use of spectroscopic (UV-vis and IR) and calorimetric techniques (heat of adsorption of water as a measure of hydrophilicity) reveals that DE/1-FC and DE/2-FC particles have an external layer of glass, absent in DE/1-C, where iron impurities act as network-forming and sodium ions as modifier species, with few patches of a hydrophobic phase, the latter relatable to a heated pure silica phase. When tested on a macrophage cell line (MH-S) in comparison with appropriate positive and negative controls (an active and an inactive quartz dust, respectively), only DE/1-C exhibited a cell damage and activation similar to that of active quartz (measured by lactate dehydrogenase release, peroxidation of membrane lipids and synthesis of NO). It is likely that the presence of a vitreous phase mitigates or even eliminates the cellular responses of silica in DE.

Classical inhibitors of NOX NAD(P)H oxidases are not specific.

NAD(P)H oxidases (NOXs) are a family of enzymes catalyzing the univalent reduction of oxygen to produce the superoxide anion radical, which in turn can be converted in other reactive oxygen species (ROS) and may participate to the formation of reactive nitrogen derivatives, such as peroxynitrite. By virtue of their activity, NOXs may represent a double-edged sword for the organism's homeostasis. On one hand ROS participate in host defence by killing invading microbes and may regulate several important physiological functions, such as cell signalling, regulation of cell growth and differentiation, oxygen sensing, angiogenesis, fertilization and control of vascular tone. On the other hand ROS may play an important role in pathological processes such as hypertension, atherosclerosis, diabetes, cancer, ischemia/reperfusion injury, neurodegenerative diseases. Many roles suggested for NOXs in various tissues and physiopathological situations have been inferred by the in vitro and in vivo effects of several NOX inhibitors. In particular, most studies are based on the use of two compounds, diphenyleneiodonium and apocynin. Aim of this review is to describe the main features of these two compounds, to show that they cannot be used as specific NOX inhibitors and to solicit researchers to find other tools for investigating the role of NOXs.

Editor's Highlight: Abrasion of Artificial Stones as a New Cause of an Ancient Disease. Physicochemical Features and Cellular Responses.

New outbursts of silicosis were recently reported among workers manufacturing an engineered material known as "artificial stone," composed by high percentages of quartz (up to 98%) agglomerated with pigments and polymeric resins. Dusts released by abrasion during artificial stone polishing were characterized for particle size, morphology, and elemental composition and studied for (1) ability to catalyze free radical generation in acellular tests, (2) membranolytic potential on human erythrocytes, (3) cytotoxic activity (lactate dehydrogenase release) on murine alveolar macrophages (MH-S) and human bronchial epithelial (BEAS-2B) cell lines, (4) induction of epithelial-mesenchymal transition (EMT) in BEAS-2B cells. Min-U-Sil 5 was used as reference quartz. Artificial stone dusts exhibited morphological features close to quartz, but contained larger amount of metal transition ions (mainly, Fe, Cu, and Ti), potentially responsible for the high reactivity in free radical generation observed. Opposite to Min-U-Sil 5, they were neither hemolytic nor cytotoxic on MH-S cells, a low cytotoxicity only being observed with BEAS-2B cells. The presence on the particle surface of residues of the resin accounts for this attenuated behavior, as hemolysis appeared and cytotoxicity increased after thermal degradation of the resin, when the free quartz surface was exposed. All dusts induced EMT with loss of E-cadherin expression and increased the expression of mesenchymal proteins (α-smooth muscle actin and vimentin). This may contribute to explain the development of fibrosis on workers exposed to artificial stone dusts.

Effects of Chrysotile Exposure in Human Bronchial Epithelial Cells: Insights into the Pathogenic Mechanisms of Asbestos-Related Diseases.

BACKGROUND: Chrysotile asbestos accounts for > 90% of the asbestos used worldwide, and exposure is associated with asbestosis (asbestos-related fibrosis) and other malignancies; however, the molecular mechanisms involved are not fully understood. A common pathogenic mechanism for these malignancies is represented by epithelial-mesenchymal transition (EMT), through which epithelial cells undergo a morphological transformation to assume a mesenchymal phenotype. In the present work, we propose that chrysotile asbestos induces EMT through a mechanism involving a signaling pathway mediated by tranforming growth factor beta (TGF-ß). OBJECTIVES: We investigated the role of chrysotile asbestos in inducing EMT in order to elucidate the molecular mechanisms involved in this event. METHODS: Human bronchial epithelial cells (BEAS-2B) were incubated with 1 µg/cm2 chrysotile asbestos for ≤ 72 hr, and several markers of EMT were investigated. Experiments with specific inhibitors for TGF-ß, glycogen synthase kinase-3ß (GSK-3ß), and Akt were performed to confirm their involvement in asbestos-induced EMT. Real-time polymerase chain reaction (PCR), Western blotting, and gelatin zymography were performed to detect mRNA and protein level changes for these markers. RESULTS: Chrysotile asbestos activated a TGF-ß-mediated signaling pathway, implicating the contributions of Akt, GSK-3ß, and SNAIL-1. The activation of this pathway in BEAS-2B cells was associated with a decrease in epithelial markers (E-cadherin and ß-catenin) and an increase in mesenchymal markers (α-smooth muscle actin, vimentin, metalloproteinases, and fibronectin). CONCLUSIONS: Our findings suggest that chrysotile asbestos induces EMT, a common event in asbestos-related diseases, at least in part by eliciting the TGF-ß-mediated Akt/GSK-3ß/SNAIL-1 pathway. CITATION: Gulino GR, Polimeni M, Prato M, Gazzano E, Kopecka J, Colombatto S, Ghigo D, Aldieri E. 2016. Effects of chrysotile exposure in human bronchial epithelial cells: insights into the pathogenic mechanisms of asbestos-related diseases. Environ Health Perspect 124:776-784; http://dx.doi.org/10.1289/ehp.1409627.

Host matrix metalloproteinases in cerebral malaria: new kids on the block against blood-brain barrier integrity?

Cerebral malaria (CM) is a life-threatening complication of falciparum malaria, associated with high mortality rates, as well as neurological impairment in surviving patients. Despite disease severity, the etiology of CM remains elusive. Interestingly, although the Plasmodium parasite is sequestered in cerebral microvessels, it does not enter the brain parenchyma: so how does Plasmodium induce neuronal dysfunction? Several independent research groups have suggested a mechanism in which increased blood-brain barrier (BBB) permeability might allow toxic molecules from the parasite or the host to enter the brain. However, the reported severity of BBB damage in CM is variable depending on the model system, ranging from mild impairment to full BBB breakdown. Moreover, the factors responsible for increased BBB permeability are still unknown. Here we review the prevailing theories on CM pathophysiology and discuss new evidence from animal and human CM models implicating BBB damage. Finally, we will review the newly-described role of matrix metalloproteinases (MMPs) and BBB integrity. MMPs comprise a family of proteolytic enzymes involved in modulating inflammatory response, disrupting tight junctions, and degrading sub-endothelial basal lamina. As such, MMPs represent potential innovative drug targets for CM.

Role of 15-hydroxyeicosatetraenoic acid in hemozoin-induced lysozyme release from human adherent monocytes.

Natural hemozoin (nHZ), a lipid-bound ferriprotoporphyrin IX crystal produced by Plasmodium parasites after hemoglobin catabolism, seriously compromises the functions of human monocytes, and 15-hydroxyeicosatetraenoic acid (15-HETE) and 4-hydroxynonenal (4-HNE), two nHZ lipoperoxidation products, have been related to such a functional impairment. nHZ was recently shown to promote inflammation-mediated lysozyme release from human monocytes through p38 mitogen-activated protein kinase- (MAPK)- and nuclear factor (NF)-κB-dependent mechanisms. This study aimed at identifying the molecule of nHZ lipid moiety that was responsible for these effects. Results showed that 15-HETE mimicked nHZ effects on lysozyme release, whereas 4-HNE did not. 15-HETE-enhanced lysozyme release was abrogated by anti-TNF-α and anti-IL-1ß-blocking antibodies and mimicked by recombinant cytokines; on the contrary, MIP-1α/CCL3 was not involved as a soluble mediator of 15-HETE effects. Moreover, 15-HETE early activated p38 MAPK and NF-κB pathways by inducing p38 MAPK phosphorylation; cytosolic I-κBα phosphorylation and degradation; NF-κB nuclear translocation and DNA-binding. Inhibition of both routes through chemical inhibitors (SB203580, quercetin, artemisinin, and parthenolide) prevented 15-HETE-dependent lysozyme release. Collectively, these data suggest that 15-HETE plays a major role in nHZ-enhanced monocyte degranulation.

Natural haemozoin induces expression and release of human monocyte tissue inhibitor of metalloproteinase-1.

Recently matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1) have been implicated in complicated malaria. In vivo, mice with cerebral malaria (CM) display high levels of both MMP-9 and TIMP-1, and in human patients TIMP-1 serum levels directly correlate with disease severity. In vitro, natural haemozoin (nHZ, malarial pigment) enhances monocyte MMP-9 expression and release. The present study analyses the effects of nHZ on TIMP-1 regulation in human adherent monocytes. nHZ induced TIMP-1 mRNA expression and protein release, and promoted TNF-α, IL-1ß, and MIP-1α/CCL3 production. Blocking antibodies or recombinant cytokines abrogated or mimicked nHZ effects on TIMP-1, respectively. p38 MAPK and NF-κB inhibitors blocked all nHZ effects on TIMP-1 and pro-inflammatory molecules. Still, total gelatinolytic activity was enhanced by nHZ despite TIMP-1 induction. Collectively, these data indicate that nHZ induces inflammation-mediated expression and release of human monocyte TIMP-1 through p38 MAPK- and NF-κB-dependent mechanisms. However, TIMP-1 induction is not sufficient to counterbalance nHZ-dependent MMP-9 enhancement. Future investigation on proteinase-independent functions of TIMP-1 (i.e. cell survival promotion and growth/differentiation inhibition) is needed to clarify the role of TIMP-1 in malaria pathogenesis.

The pentose phosphate pathway: an antioxidant defense and a crossroad in tumor cell fate.

The pentose phosphate pathway, one of the main antioxidant cellular defense systems, has been related for a long time almost exclusively to its role as a provider of reducing power and ribose phosphate to the cell. In addition to this "traditional" correlation, in the past years multiple roles have emerged for this metabolic cascade, involving the cell cycle, apoptosis, differentiation, motility, angiogenesis, and the response to anti-tumor therapy. These findings make the pentose phosphate pathway a very interesting target in tumor cells. This review summarizes the latest discoveries relating the activity of the pentose phosphate pathway to various aspects of tumor metabolism, such as cell proliferation and death, tissue invasion, angiogenesis, and resistance to therapy, and discusses the possibility that drugs modulating the pathway could be used as potential tools in tumor therapy.

Haemozoin induces early cytokine-mediated lysozyme release from human monocytes through p38 MAPK- and NF-kappaB-dependent mechanisms.

Malarial pigment (natural haemozoin, HZ) is a ferriprotoporphyrin IX crystal produced by Plasmodium parasites after haemoglobin catabolism. HZ-fed human monocytes are functionally compromised, releasing increased amounts of pro-inflammatory molecules, including cytokines, chemokines and cytokine-related proteolytic enzyme Matrix Metalloproteinase-9 (MMP-9), whose role in complicated malaria has been recently suggested. In a previous work HZ was shown to induce through TNFalpha production the release of monocytic lysozyme, an enzyme stored in gelatinase granules with MMP-9. Here, the underlying mechanisms were investigated. Results showed that HZ lipid moiety promoted early but not late lysozyme release. HZ-dependent lysozyme induction was abrogated by anti-TNFalpha/IL-1 beta/MIP-1 alpha blocking antibodies and mimicked by recombinant cytokines. Moreover, HZ early activated either p38 MAPK or NF-kappaB pathways by inducing: p38 MAPK phosphorylation; cytosolic I-kappaB alpha phosphorylation and degradation; NF-kappaB nuclear translocation and DNA-binding. Inhibition of both routes through selected molecules (SB203580, quercetin, artemisinin, parthenolide) prevented HZ-dependent lysozyme release. These data suggest that HZ-triggered overproduction of TNFalpha, IL-1 beta and MIP-1 alpha mediates induction of lysozyme release from human monocytes through activation of p38 MAPK and NF-kappaB pathways, providing new evidence on mechanisms underlying the HZ-enhanced monocyte degranulation in falciparum malaria and the potential role for lysozyme as a new affordable marker in severe malaria.

Physicochemical determinants in the cellular responses to nanostructured amorphous silicas.

Amorphous silicas, opposite to crystalline polymorphs, have been regarded so far as nonpathogenic, but few studies have addressed the toxicity of the wide array of amorphous silica forms. With the advent of nanotoxicology, there has been a rising concern about the safety of silica nanoparticles to be used in nanomedicine. Here, we report a study on the toxicity of amorphous nanostructured silicas obtained with two different preparation procedures (pyrolysis vs. precipitation), the pyrogenic in two very different particle sizes, in order to assess the role of size and origin on surface properties and on the cell damage, oxidative stress, and inflammatory response elicited in murine alveolar macrophages. A quartz dust was employed as positive control and monodispersed silica spheres as negative control. Pyrogenic silicas were remarkably more active than the precipitated one as to cytotoxicity, reactive oxygen species production, lipid peroxidation, nitric oxide synthesis, and production of tumor necrosis factor-α, when compared both per mass and per unit surface. Between the two pyrogenic silicas, the larger one was the more active. Silanols density is the major difference in surface composition among the three silicas, being much larger than the precipitated one as indicated by joint calorimetric and infrared spectroscopy analysis. We assume here that full hydroxylation of a silica surface, with consequent stable coverage by water molecules, reduces/inhibits toxic behavior. The preparation route appears thus determinant in yielding potentially toxic materials, although the smallest size does not always correspond to an increased toxicity.

Macrophage inflammatory protein-1alpha mediates matrix metalloproteinase-9 enhancement in human adherent monocytes fed with malarial pigment.

OBJECTIVE: To investigate the role of macrophage inflammatory protein-1alpha (MIP-1alpha) in the detrimental enhancement of matrix metalloproteinase-9 (MMP-9) expression, release and activity induced by phagocytosis of malarial pigment (haemozoin, HZ) in human monocytes. METHODS: Human adherent monocytes were unfed/fed with native HZ for 2 h. After 24 hours, MIP-1alpha production was evaluated by ELISA in cell supernatants. Alternatively, HZ-unfed/fed monocytes were treated in presence/absence of anti-human MIP-1alpha blocking antibodies or recombinant human MIP-1alpha for 15 h (RNA studies) or 24 h (protein studies); therefore, MMP-9 mRNA expression was evaluated in cell lysates by Real Time RT-PCR, whereas proMMP-9 and active MMP-9 protein release were measured in cell supernatants by Western blotting and gelatin zymography. RESULTS: Phagocytosis of HZ by human monocytes increased production of MIP-1 alpha, mRNA expression of MMP-9 and protein release of proMMP-9 and active MMP-9. All the HZ-enhancing effects on MMP-9 were abrogated by anti-human MIP-1alpha blocking antibodies and mimicked by recombinant human MIP-1alpha. CONCLUSIONS: The present work suggests a role for MIP-1alpha in the HZ-dependent enhancement of MMP-9 expression, release and activity observed in human monocytes, highlighting new detrimental effects of HZ-triggered proinflammatory response by phagocytic cells in falciparum malaria.

Quartz inhibits glucose 6-phosphate dehydrogenase in murine alveolar macrophages.

Crystalline silica is well-known to induce oxidative stress as a consequence of both surface-derived generation of free radicals and intracellular production of reactive oxygen species upon phagocytosis; the mechanism of the latter is still partially unknown. In this study, we report that in murine alveolar MH-S macrophages, a 24 h incubation with quartz particles (80 microg/cm(2)) inhibits the glucose 6-phosphate dehydrogenase (G6PD) (1) activity by 70% and the pentose phosphate pathway by 30%. Such effects are accompanied by a 50% decrease of intracellular glutathione, a 35% increase of thiobarbituric acid reactive products (index of lipoperoxidation), and a 5-fold increase of leakage of lactate dehydrogenase in the extracellular medium (index of cytotoxicity). Quartz inhibits G6PD but not other oxidoreductases, and such inhibition is fully prevented by glutathione, suggesting that silica exerts on G6PD an oxidative damage. Our data provide a new additional mechanism by which silica may induce oxidative stress, that is, by inhibiting the pentose phosphate pathway, one of the main antioxidant metabolic pathways of the cell.

The NADPH oxidase inhibitor apocynin induces nitric oxide synthesis via oxidative stress.

We have recently shown that apocynin elicits an oxidative stress in N11 mouse glial cells and other cell types. Here we report that apocynin increased the accumulation of nitrite, the stable derivative of nitric oxide (NO), in the extracellular medium of N11 cell cultures, and the NO synthase (NOS) activity in cell lysates. The increased synthesis of NO was associated with increased expression of inducible NOS (iNOS) mRNA, increased nuclear translocation of the redox-sensitive transcription factor NF-kappa B and decreased intracellular level of its inhibitor IkB alpha. These effects, accompanied by increased production of H2O2, were very similar to those observed after incubation with bacterial lipopolysaccharide (LPS) and were inhibited by catalase. These results suggest that apocynin, similarly to LPS, induces increased NO synthesis by eliciting a generation of reactive oxygen species (ROS), which in turn causes NF-kappa B activation and increased expression of iNOS. Therefore, the increased bioavailability of NO reported in the literature after in vivo or in vitro treatments with apocynin might depend, at least partly, on the drug-elicited induction of iNOS, and not only on the inhibition of NADPH oxidase and the subsequent decreased scavenging of NO by oxidase-derived ROS, as it is often supposed.