Ζ potential evidences silanol heterogeneity induced by metal contaminants at the quartz surface: Implications in membrane damage.

Among the physico-chemical features responsible for the so-called "variability of quartz hazard", a key role has been assigned to the silica surface charge, evaluated by means of ζ potential measurement. The ζ potential of silica describes the protonation state of silanols which, in turn, determine interactions with cell membranes. To gain a molecular understanding of the role of silanols in silica pathogenicity, we conducted a systematic investigation of the variation of the ζ potential as a function of pH (ζ plot titration curve) on a large set of respirable quartz particles with different levels of metal contaminants. The membranolytic activity of the particles on red blood cells, used as a readout of pathogenic activity, was assessed in parallel. Pure quartz surfaces showed sigmoid-shaped ζ plots suggesting the presence of silanol families with similar acidity, whereas contaminated dusts exhibited convex-shaped ζ plots, indicating a higher silanol heterogeneity on contaminated surfaces with respect to the pure ones. The quartz particles with a higher surface heterogeneity related to metal contamination showed a higher membranolytic activity. By removing structural defects and chemical heterogeneity, the ζ plot shifted towards the typical shape of pure quartz and the membranolytic activity was reduced. We conclude that the ζ plot is a useful readout to measure the acid-base behavior of quartz surfaces and to describe the chemical heterogeneity of quartz silanols. Surface heterogeneity, here induced by metal contamination, is proposed as the main cause of quartz membranolytic activity, further supporting the hypothesis that surface silanol disorganization determines silica pathogenicity.

In search of the chemical basis of the hemolytic potential of silicas.

The membranolytic activity of silica particles toward red blood cells (RBCs) has been known for a long time and is sometimes associated with silica pathogenicity. However, the molecular mechanism and the reasons why hemolysis differs according to the silica form are still obscure. A panel of 15 crystalline (pure and commercial) and amorphous (pyrogenic, precipitated from aqueous solutions, vitreous) silica samples differing in size, origin, morphology, and surface chemical composition were selected and specifically prepared. Silica particles were grouped into six groups to compare their potential in disrupting RBC membranes so that one single property differed in each group, while other features were constant. Free radical production and crystallinity were not strict determinants of hemolytic activity. Particle curvature and morphology modulated the hemolytic effect, but silanols and siloxane bridges at the surface were the main actors. Hemolysis was unrelated to the overall concentration of silanols as fully rehydrated surfaces (such as those obtained from aqueous solution) were inert, and one pyrogenic silica also lost its membranolytic potential upon progressive dehydration. Overall results are consistent with a model whereby hemolysis is determined by a defined surface distribution of dissociated/undissociated silanols and siloxane groups strongly interacting with specific epitopes on the RBC membrane.

Singlet oxygen plays a key role in the toxicity and DNA damage caused by nanometric TiO2 in human keratinocytes.

Nanometric TiO2 has been reported to be cytotoxic and genotoxic in different in vitro models when activated by UV light. However, a clear picture of the species mediating the observed toxic effects is still missing. Here, a nanometric TiO2 powder has been modified at the surface to completely inhibit its photo-catalytic activity and to inhibit the generation of all reactive species except for singlet oxygen. The prepared powders have been tested for their ability to induce strand breaks in plasmid DNA and for their cytotoxicity and genotoxicity toward human keratinocyte (HaCaT) cells (100-500 µg mL(-1), 15 min UVA/B exposure at 216-36 mJ m(-2) respectively). The data reported herein indicate that the photo-toxicity of TiO2 is mainly triggered by particle-derived singlet oxygen. The data presented herein contribute to the knowledge of structure-activity relationships which are needed for the design of safe nanomaterials.

Carbon in intimate contact with quartz reduces the biological activity of crystalline silica dusts.

To evaluate the effect of carbonaceous materials on the pathogenic activity of quartz dusts, mixtures of carbon soot (1 and 10%) and quartz (Min-U-Sil) were prepared and then milled so to attain an intimate association of carbon and the quartz surface. Both cellular and cell-free tests show that carbon associated to quartz completely inhibits the typical free radical generation of quartz dusts (through Fenton activity and homolytic cleavage of a C-H bond) and suppresses the oxidative stress and inflammation induced by quartz alone on MH-S murine macrophage cells (lipid peroxidation, nitric oxide release, and tumor necrosis factor-α synthesis). The cytotoxic response to quartz is also largely reduced. An extremely pure quartz milled with 10% of soot showed inactivating effects on the adverse reactions to quartz similar to Min-U-Sil quartz. None of these effects takes place when the same experiments are carried out with mechanically mixed samples, which suggests that carbon acts not just as a radical quencher but because of its association to the quartz surface.

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.

Hematite nanoparticles larger than 90 nm show no sign of toxicity in terms of lactate dehydrogenase release, nitric oxide generation, apoptosis, and comet assay in murine alveolar macrophages and human lung epithelial cells.

Three hematite samples were synthesized by precipitation from a FeCl3 solution under controlled pH and temperature conditions in different morphology and dimensions: (i) microsized (average diameter 1.2 µm); (ii) submicrosized (250 nm); and (iii) nanosized (90 nm). To gain insight into reactions potentially occurring in vivo at the particle-lung interface following dust inhalation, several physicochemical features relevant to pathogenicity were measured (free radical generation in cell-free tests, metal release, and antioxidant depletion), and cellular toxicity assays on human lung epithelial cells (A549) and murine alveolar macrophages (MH-S) were carried out (LDH release, apoptosis detection, DNA damage, and nitric oxide synthesis). The decrease in particles size, from 1.2 µm to 90 nm, only caused a slight increase in structural defects (disorder of the hematite phase and the presence of surface ferrous ions) without enhancing surface reactivity or cellular responses in the concentration range between 20 and 100 µg cm⁻².

Model system to study the influence of aggregation on the hemolytic potential of silica nanoparticles.

A well-defined silica nanoparticle model system was developed to study the effect of the size and structure of aggregates on their membranolytic activity. The aggregates were stable and characterized using transmission electron microscopy, dynamic light scattering, nitrogen adsorption, small-angle X-ray scattering, infrared spectroscopy, and electron paramagnetic resonance. Human red blood cells were used for assessing the membranolytic activity of aggregates. We found a decreasing hemolytic activity for increasing hydrodynamic diameter of the nanoparticle aggregates, in contrast to trends observed for isolated particles. We propose here a qualitative model that considers the fractal structure of the aggregates and its influence on membrane deformation to explain these observations. The open structure of the aggregates means that only a limited number of primary particles, from which the aggregates are built up, are in contact with the cell membrane. The adhesion energy is thus expected to decrease resulting in an overall lowered driving force for membrane deformation. Hence, the hemolytic activity of aggregates, following an excessive deformation of the cell membrane, decreases as the aggregate size increases. Our results indicate that the aggregate size and structure determine the hemolytic activity of silica nanoparticle aggregates.

Interaction of spherical silica nanoparticles with neuronal cells: size-dependent toxicity and perturbation of calcium homeostasis.

The effects of Stöber silica nanoparticles on neuronal survival, proliferation, and on the underlying perturbations in calcium homeostasis are investigated on the well-differentiated neuronal cell line GT1-7. The responses to nanoparticles 50 and 200 nm in diameter are compared. The 50-nm silica affects neuronal survival/proliferation in a dose-dependent way, by stimulating apoptotic processes. In contrast, the 200-nm silica does not show any toxic effect even at relatively high concentrations (292 µg mL−1). To identify the mechanisms underlying these effects, the changes in intracellular calcium concentration elicited by acute and chronic administration of the two silica nanoparticles are analyzed. The 50-nm silica at toxic concentrations generates huge and long-lasting increases in intracellular calcium, whereas the 200-nm silica only induces transient signals of much lower amplitude. These findings provide the first evidence that silica nanoparticles can induce toxic effects on neuronal cells in a size-dependent way, and that these effects are related to the degree of perturbation of calcium homeostasis.

Surface iron inhibits quartz-induced cytotoxic and inflammatory responses in alveolar macrophages.

The mechanism of enhancement/inhibition of quartz toxicity induced by iron is still unclear. Here the amount of iron on a fibrogenic quartz (Qz) was increased by wet impregnation (Fe(NO(3))(3) 0.67 and 6.7 wt %). X-ray diffraction (XRD), XRF diffuse reflectance, UV-vis, and infrared (IR) spectroscopies revealed dispersed ferric ions, and hematite aggregates at the higher loading. Surface features relevant to pathogenicity and cell responses were compared not only to the original quartz but also to reference quartz DQ12. Surface charge (ζ-potential) was more negative on the original and low-loaded specimen than on the high-loaded one. DQ12 had a less negative ζ-potential than Qz, ascribed to the absence of aluminium present in Qz (1.7 wt %). All quartz specimens were able to generate HO(•) radicals, iron-loaded samples being more reactive than original quartz. Iron deposition inhibited the rupture of a C-H bond. All quartzes were phagocytized by alveolar macrophages (AMΦ cell line NR8383) to the same extent, irrespective of their surface state. Conversely, iron loading increased AMΦ viability (evaluated by cytotoxicity and induction of apoptosis). Qz was found to be much less cytotoxic than DQ12. The induction of oxidative stress and inflammatory responses (evaluated by HO-1 mRNA expression and TNF-α mRNA and protein expression) revealed a reduction in inflammogenicity upon iron loading and a more inflammogenic potency of DQ12 ascribed to undissociated SiOH interacting via H-bonding with cell membrane components. The results suggest that besides aluminium also iron at the quartz surface may have an inhibitory effect on adverse health responses.

Physico-chemical features of engineered nanoparticles relevant to their toxicity.

Nanotoxicology studies require investigations of several physico-chemical aspects of the particle/body fluid interaction, here described by reviewing recent literature in the light of new experimental data. Current characterization mostly covers morphology and metric-related characteristics (form, chemical composition, specific surface area, primary particle size and size distribution), and is mandatory in any experimental study. To unveil toxicity mechanisms, several other physico-chemical properties relevant to (geno) toxicity need to be assessed, typically the release or quenching of radical/ROS (Reactive Oxygen Species), the presence of active metal ions, evidence of structural defects. Major tasks for physical chemists working on nanoparticles-induced genotoxicity are described with some examples: (i), Tailored preparation of the same material in different sizes; (ii) particle modification changing a single property at a time; and (iii) identification of appropriate reference materials. Phenomena occurring during the contact between nanoparticles and cellular media or biological fluids (dispersion, agglomeration/aggregation, protein adsorption) are discussed in relation to the surface properties of the nanoparticles considered.

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.

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.

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.

Surface reactivity, cytotoxic, and morphological transforming effects of diatomaceous Earth products in Syrian hamster embryo cells.

In order to evaluate the effect of thermal treatments on the surface reactivity and carcinogenic potential of diatomaceous earth (DE) products, the physicochemical features of some specimens--derived by heating the same original material--were compared with their cytotoxic and transforming potency. The samples were an untreated DE (amorphous) progressively heated in the laboratory at 900 degrees C (DE 900) and 1200 degrees C (DE 1200) and a commercial product manufactured from the same DE (Chd) from which the finer fraction (< 10-microm diameter) was separated (Chd-F). Quartz (Min-U-Sil 5) and a vitreous silica (amorphous) smoothed up with hydrofluoric acid and were used as positive and negative controls, respectively. All samples were analyzed for their degree of crystallization, for their ability to release free radicals and reactive oxygen species, and for their cytotoxic and transforming potencies in Syrian hamster embryo (SHE) cells. X-ray diffractometry showed that DE 900, like DE, was still amorphous, whereas DE 1200 as well as the commercial product (Chd) were partially crystallized into cristobalite. The ability of the dust to release hydroxyl (*OH) radicals in the presence of hydrogen peroxide, as revealed by the spin-trapping technique, was as follows: Chd-F, DE 1200 > Chd > DE 900 > DE, suggesting that on heating, the surface acquires a higher potential for free radical release. Most of the silica samples generated COO* radicals from the formate ion, following homolytic rupture of the carbon-hydrogen bond, in the presence of ascorbic acid. A concentration-dependent decrease in cell proliferation and colony-forming efficiency was observed in SHE cultures treated with Chd-F, Chd, and DE. Heating abolished DE cytotoxicity but conferred a transforming ability to thermal treated particles. DE was the only sample that did not induce morphological transformation of cells. According to their transformation capacity, the samples were classified as follows: Chd-F > Chd, DE 1200 > DE 900 >> DE. Taken together, the reported results suggest that (1) the transforming potential of a biogenic amorphous silica is related to the thermal treatment that transforms the original structure in cristobalite and generates surface active sites; (2) the reactivity of samples in releasing *OH radicals correlates to their transforming ability; (3) the finer fraction of the commercial product is significantly more toxic and transforming than the coarse dust; and (4) opposite to silica dusts of mineral origin, which loose both cytotoxicity and transforming ability upon heating, heated diatomite acquires a cell-transforming potency. DE products should be thus considered a set apart of silica-based potentially toxic materials.

Relationship between the state of the surface of four commercial quartz flours and their biological activity in vitro and in vivo.

Four commercial quartz dusts (flours), two inflammogenic in vivo and activating macrophages in vitro (Qz 2/1-c and Qz 3/1-c) and two mostly inert (Qz 5/1-c and Qz 11/1-c), have been compared regarding their surface properties, in order to detect chemical differences which may account for their different biological behaviour. The following features have been examined: 1) extent of the amorphous fraction (heat associated alpha<-->beta transition of quartz) and its solubility in HF; 2) potential to cleave a carbon-hydrogen bond with consequent generation of carbon centred radicals (spin trapping technique, EPR); 3) evolution of surface functionalities upon heating (FTIR spectroscopy); 4) mechanisms of adsorption of water on dusts outgassed at 150 degrees and at 800 degrees C (adsorption calorimetry). HCl treated samples have also been examined. The two "less toxic" quartzes are more resistant to HF attack, coordinate irreversibly H2O molecules and exhibit strong adsorption sites, which are absent in the other two and in a very pure quartz dust. Conversely all samples show the same potential to release free radicals. The different behaviour of the two sets of dust is consistent with a different level of impurities, namely aluminium ex kaolin, carbon and alkaline ions. The less inflammogenic quartzes appear to be covered by aluminium ions (and possibly iron) which strongly holds molecular water or carbonates, thus reducing the silanol patches to a large extent and changing the surface properties of the particles. We hypothesize that cellular response, and particularly macrophage activation and death, is mediated by strong interactions between silanol patches and some cell membrane components, but inhibited when the surface of the particle is modified by the presence of aluminium ions, surface carbonates and other metal contaminants. This hypothesis suggests that grinding procedures with little appropriate additives, e.g. kaolin, alumina, can reduce the biological activity of quartz dusts.

Crystalline silica incubated in ascorbic acid acquires a higher cytotoxic potential.

Quartz incubated in an aqueous solution of ascorbic acid is partially dissolved and the potential to generate hydroxyl radicals from hydrogen peroxide is enhanced. In order to investigate whether the surface activation triggered by the treatment with ascorbic acid would also involve an enhancement in cell toxicity, a murine macrophage cell line (RAW 264.7) was exposed to untreated and ascorbic acid-treated quartz. Ascorbic acid pretreated quartz was more toxic than untreated quartz and all cells died within 24 hours after exposure. Tetrandrine (a Chinese drug employed to retard or reverse fibrotic lesions of silicosis in humans) partially reduced cell toxicity generated by ascorbic acid pretreated quartz.