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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.

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.

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.

"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.

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.

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 1200ChdDE 900DE, 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-FChd, DE 1200DE 900DE. 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.

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.