Food-grade titanium dioxide can affect microbiota physiology, adhesion capability, and interbacterial interactions: A study on L. rhamnosus and E. faecium.

Food-grade titanium dioxide (TiO2-FG) is a widespread metal oxide used in the food industries. Recently, the European Food Safety Authority concluded that TiO2-FG cannot be considered safe for consumption due to its genotoxicity; however, its effect on the gut microbiota has not yet been completely unraveled. We studied the effects of TiO2-FG (0.125 mg/mL) on Lactobacillus rhamnosus GG (LGG) and Enterococcus faecium NCIMB10415 (Ent), in particular some physiological and phenotypic traits (growth kinetics, bile salts, and ampicillin resistance) and their interactions with the host (auto-aggregation, biofilm formation, and adhesion on Caco-2/TC7 monolayers) and other gut microorganisms (antimicrobial activity towards pathogens). The results obtained revealed that TiO2-FG alters both LGG and Ent growth and lowers bile resistance (62 and 34.5%, respectively) and adhesion on Caco-2/TC7 monolayers (34.8 and 14.16%, respectively). The other outcomes were strictly species-specific: Ent showed a lower ampicillin sensitivity (14.48%) and auto-aggregation (38.1%), while LGG showed a reduced biofilm formation (37%) and antimicrobial activity towards Staphylococcus aureus (35.73%). Overall, these results suggest an adverse effect of TiO2-FG on both the endogenous and exogenously administered probiotics, contributing to the argument against using TiO2-FG as a food additive.

The role of iron impurities in the toxic effects exerted by short multiwalled carbon nanotubes (MWCNT) in murine alveolar macrophages.

Lung toxicity mediated by multiwalled carbon nanotubes (MWCNT) has been widely demonstrated and recently associated with induction of carcinogenic asbestos-like effects, but the chemical features that drive this toxic effect have still not been well elucidated. The presence of metals as trace contaminants during MWCNT preparation, in particular iron (Fe) impurities, plays an important role in determining a different cellular response to MWCNT. Our goal was to clarify the mechanisms underlying MWCNT-induced toxicity with correlation to the presence of Fe impurities by exposing murine alveolar macrophages to two different MWCNT samples, which differed only in the presence or absence of Fe. Data showed that only Fe-rich MWCNT were significantly cytotoxic and genotoxic and induced a potent cellular oxidative stress, while Fe-free MWCNT did not exert any of these adverse effects. These results confirm that Fe content represents an important key constituent in promoting MWCNT-induced toxicity, and this needs to be taken into consideration when planning new, safer preparation routes.

Hydrophilic/hydrophobic features of TiO2 nanoparticles as a function of crystal phase, surface area and coating, in relation to their potential toxicity in peripheral nervous system.

The hydrophilic/hydrophobic properties of a variety of commercial TiO(2) nanoparticles (NP), to be employed as inorganic filters in sunscreen lotions, were investigated both as such (dry powders) and dispersed in aqueous media. Water uptake and the related interaction energy have been determined by means of adsorption microcalorimetry of H(2)O vapor, whereas dispersion features in aqueous solutions were investigated by dynamic light scattering and electrokinetic measurements (zeta potential). The optimized dispersions in cell culture medium were employed to assess the possible in vitro neuro-toxicological effect on dorsal root ganglion (DRG) cells upon exposure to TiO(2)-NP, as a function of crystal phase, surface area and coating. All investigated materials, with the only exception of the uncoated rutile, were found to induce apoptosis on DRG cells; the inorganic/organic surface coating was found not to protect against the TiO(2)-induced apoptosis. The risk profile for DRG cells, which varies for the uncoated samples in the same sequence as the photo-catalytic activity of the different polymorphs: anatase-rutile>anatase>>rutile, was found not to be correlated with the surface hydrophilicity of the uncoated/coated specimens. Aggregates/agglomerates hydrodynamic diameter was comprised in the ~200-400 nm range, compatible with the internalization within DRG cells.

Free radical generation in the toxicity of inhaled mineral particles: the role of iron speciation at the surface of asbestos and silica.

Free radical generation at the particle/biological fluid interface is one of the chemical processes that contributes to pathogenicity. In order to investigate the role played by iron, fibres of crocidolite asbestos have been modified by thermal treatments to alter their surface iron content. Two radical mechanisms, HO* from H2O2 and cleavage of a C-H bond, which are both active on the original fibres, have been tested on the modified fibres. C-H cleavage is dependent on Fe(II) abundance and location and is suppressed by surface oxidation while HO* release appears independent of the oxidation state of iron. Quartz specimens with different levels of iron impurities have been tested in a similar manner. A commercially available quartz (Min-U-Sil 5) containing trace levels of iron is also active in both tests, but reactivity is not fully suppressed by treatment with desferrioxamine, which should remove/inactivate iron. The radical yield attained is close to the level produced by a pure quartz dust, suggesting the presence of active sites other than iron. Ascorbic acid reacts with both crocidolite and quartz, with subsequent depletion of the level of antioxidant defences when particle deposition occurs in the lung lining layer. Following treatment with ascorbic acid the radical yield increases with quartz, but decreases with asbestos. Selective removal of iron and silicon from the surface may account for the differences in behaviour of the two particulates.

Variability of biological responses to silicas: effect of origin, crystallinity, and state of surface on generation of reactive oxygen species and morphological transformation of mammalian cells.

Variously modified quartz dusts and one amorphous diatomaceous earth have been compared in their potential to release HO* radicals and in their activity in the Syrian hamster embryo (SHE) cell transformation assay. Both original dusts, made up by well-crystallized quartz particles, or by mostly amorphous, variously shaped, silica particles, were active in HO* release, were cytotoxic, and induced morphological transformation in SHE cells. The cristobalite dust, obtained by heating quartz above the phase transition temperature, lost any activity in free radical release, cytotoxicity, and transforming potency. Surface-modified quartz dusts were obtained by a mild etching with HF, by depriving the surface of trace iron with deferoxamine, or by enriching it with iron. The chemical and biological activity decreased in all cases. Both iron-deprived and iron-enriched quartz were nearly inactive. A linear correlation was found between the amount of HO* released by the particles and the transformation frequency. When the SHE cell assay was performed in the presence of mannitol or antioxidant enzymes (superoxide dismutase [SOD] or catalase), the number of transformed cells markedly decreased. This effect was more pronounced for catalase and mannitol than for SOD. HO* release was reduced, but not suppressed, by deferoxamine. All the above results are consistent with the presence of two kinds of surface sites active in HO* release and cell transformation: (1) silicon-based radicals, abundant on freshly ground dusts, which generate the HO* radicals without the superoxide ion as intermediate; and (2) isolated iron centers where the Haber-Weiss cycle takes place, with the superoxide ion as intermediate. The activities of both sites are inhibited by mannitol or catalase, whereas only the last one is inhibited by SOD.

Silica and renal diseases: no longer a problem in the 21st century?

Silicosis and other occupational diseases are still important even in the most developed countries. In fact, at present, silica exposure may be a risk factor for human health not only for workers but also for consumers. Furthermore, this exposure is associated with many other different disorders besides pulmonary silicosis, such as progressive systemic sclerosis, systemic lupus erythematosus, rheumatoid arthritis, dermatomyositis, glomerulonephritis and vasculitis. The relationships between these silica-related diseases need to be clarified, but pathogenic responses to silica are likely to be mediated by interaction of silica particles with the immune system, mainly by activation of macrophages. As regards renal pathology, there is no single specific clinical or laboratory finding of silica-induced nephropathy: renal involvement may occur as a toxic effect or in a context of autoimmune disease, and silica damage may act as an additive factor on an existing, well-established renal disease. An occupational history must be obtained for all renal patients, checking particularly for exposure to silica, heavy metals, and solvents.

Iron inhibits the nitric oxide synthesis elicited by asbestos in murine macrophages.

Crocidolite fibers stimulated nitric oxide synthase (NOS) activity and expression in glial and alveolar murine macrophages: this effect was inhibited by iron supplementation and enhanced by iron chelation. We suggest that in these cells crocidolite stimulates NOS expression by decreasing the iron bioavailability and activating an iron-sensitive transcription factor.

Free radical activity of natural and heat treated amphibole asbestos.

The amphibole minerals amosite and crocidolite were subjected to calcination and to hydrothermal treatment in order to study the effect of these heat treatments on the ability of the minerals to trigger formation of free radicals, which is known to be a main factor causing asbestosis and other asbestos-induced diseases. Free radical activity of the natural and heat treated minerals was studied by using supercoiled DNA (pUC18 plasmid) as a target molecule, and also by means of EPR spectroscopy. It was shown that after calcination of the natural minerals at 1073 K their free radical activity was strongly decreased These results, which may have relevant consequences for asbestos technology, were correlated with concomitant alteration of the structure and surface chemistry of the minerals during calcination.

Spontaneous polymerisation on amphibole asbestos: relevance to asbestos removal.

Taking advantage of the spontaneous polymerisation of eugenol to lignin-like species catalysed by the surface of crocidolite fibres, a procedure is proposed, possibly useful in asbestos removal and disposal, where the polymer avoids the release of airborne fibres and also scavenges ROS (reactive oxygen species).

Possible role of ascorbic acid in the oxidative damage induced by inhaled crystalline silica particles.

The selective interaction of ascorbic acid with crystalline silica (quartz) has been studied by measuring the ascorbic acid consumption (by means of UV/vis and IR spectroscopy) and the release of silicon when quartz particles or amorphous silica (Aerosil 50) is incubated in ascorbic acid solution. At a physiological ascorbic acid concentration, quartz, and not amorphous silica, reacts, suggesting the formation of a 1:1 silicon-ascorbate complex, while at higher concentrations, the reacting amount of ascorbic acid exceeds the amount of silicon that is released. Silicon tetrahedra bearing free silanols at the quartz surface are selectively attached by ascorbic acid. The particle-derived hydroxyl radical yield in the presence of hydrogen peroxide is increased on ascorbic acid-treated quartz in comparison with the original sample. The results presented herein are relevant because the depletion of ascorbic acid from the lung lining layer and the increased potential in particle-derived free radical generation may both contribute to the oxidative damage following inhalation of crystalline silica.

Cytotoxic and transforming effects of silica particles with different surface properties in Syrian hamster embryo (SHE) cells.

Several crystalline and amorphous silica dusts (two quartz of natural origin, one cristobalite of natural and two of biogenic origin, three amorphous diatomite earths and one pyrogenic amorphous silica) were studied in the SHE cell transformation assay, in order to compare their cytotoxic and transforming potencies and examine the role of the structure and of the state of the surface on these effects. Some samples were modified by grinding, etching and heating with the aim of establishing relationships between single surface properties and biological responses. The results showed that some quartz and cristobalite dusts (crystalline) as well as the diatomaceous earths (amorphous), but not the pyrogenic amorphous silica, were cytotoxic and induced morphological transformation of SHE cells in a concentration-dependent manner. The ranking in cytotoxicity was different from that in transforming potency, suggesting two separate molecular mechanisms for the two effects. The cytotoxic and transforming potencies were different from one dust to another, even among the same structural silicas. The type of crystalline structure (quartz vs cristobalite) and the crystalline vs biogenic amorphous form did not correlate with cytotoxic or transforming potency of silica dusts. Comparison of cellular effects induced by original and surface modified samples revealed that several surface functionalities modulate cytotoxic and transforming potencies. The cytotoxic effects appeared to be related to the distribution and abundance of silanol groups and to the presence of trace amounts of iron on the silica surface. Silica particles with fractured surfaces and/or iron-active sites, able to generate reactive oxygen species, induced SHE cell transformation. The results show that the activity of silica at the cellular level is sensitive to the composition and structure of surface functionalities and confirm that the biological response to silica is a surface originated phenomenon.

Pure-silica zeolites (Porosils) as model solids for the evaluation of the physicochemical features determining silica toxicity to macrophages.

The interaction between inhaled particles and alveolar macrophages plays a key role in silica-related diseases. It has been previously shown [Fubini, B., et al. (1999) Chem. Res. Toxicol. 12, 737-745] that a monocyte-macrophage cell line (J774) may be employed in the evaluation of the degree of cytotoxicity to alveolar macrophages of various silica dusts. In this paper, pure-silica zeolites (porosils) in microcrystalline form have been employed as "model solids" in an effort to show which physicochemical properties of the silica particle are playing a major role in the toxicity to macrophages. The samples employed covered four different porosil crystal structures (MFI, FAU, TON, and MTT) and also include a synthetic rodlike cristobalite (CRIS-rd). When compared at equal weight, the samples cover a wide range of cytotoxicity from inert to toxic as unheated mineral cristobalite [Fubini, B., et al. (1999) Chem. Res. Toxicol. 12, 737-745]. Mild grinding did not affect cytotoxicity. Calcined (open pores) and uncalcined (pore filled with template) TON exhibited the same cytotoxicity, indicating that only the outer surface is implied. The hydrophobic and/or hydrophilic character of TON, evaluated by adsorption calorimetry, is close to what has been previously found for silicalite and is consistent with a hydrophilic outer surface and hydrophobic pore walls. The potential for generating hydroxyl radicals from hydrogen peroxide varies among the various porosils that have been studied. A model is proposed for the correlation between inhibition of growth on proliferating cells and physicochemical properties varying from one to the other sample. The extent of external surface and the aspect ratio were related to the intensity of the cytotoxic effect, while the level of radical release was not. This suggests, on one hand, that comparison of toxicity among various dusts should be made at equal particle surface and, on the other, that in the model studied, free radical release does not play a crucial role in the primary event of toxicity to alveolar macrophages.

Effect of Micromorphology and Surface Reactivity of Several Unusual forms of Crystalline Silica on the toxicity to a Monocyte-Macrophage Tumor Cell Line.

The fibrogenic or carcinogenic response to the inhalation of crystalline silica dusts is strictly related to the physicochemical properties of the particles, which, in turn, are mostly determined by the "origin" and the history of the dust. Several physicochemical properties have been reported to modulate silica pathogenicity. None of them simply correlate with the reported toxicity in all the systems used to study silica pathogenicity. This confirms, on the one hand, that several properties are implicated at the same time, and on the other that pathogenicity is the result of a multistage process. There is a general consensus on the key role played by alveolar macrophages in silica-related diseases. For this article the cytotoxicity of a large variety of silicas, including rather unusual forms, with controlled micromorphology and surface properties, has been studied on a mouse monocyte-machrophage tumor cell line successfully employed in previous studies on cristobalite (Fubini et al., 1999). When compared on a per unit surface basis, crystalline silicas were more cytotoxic than amorphous ones, with the notable exception of stishovite, the nonpathogenic crystalline polymorph, with octahedrally coordinated silicon atoms. Among the amorphous ones, a diatomaceous earth and a powdered silica glass exhibited an intermediate toxicity, higher than what was elicited by a pyrogenic silica. In this study a new class of crystalline silicas have been considered, pure-silica zeolites, which constitute a new morphological entity with which cells may be confronted. The cytotoxicity of these samples varies from inert to highly cytotoxic, covering all the range of toxicity covered by the traditional silica dusts. We discuss the influence of morphological properties and surface reactivity on the cytotoxicity of several pure-silica zeolites. The extent of exposed surface and the shape of the particles correlate with cell toxicity. The lower cytotoxicity of one "non-pathogenic quartz" and of an aluminum-coated Min-U-Sil quartz, compared with the original pathogenic Min-U-Sil quartz, suggest a depressive effect of the aluminum ions present at the surface of both quartzes. The extreme variability in the biological response to crystalline silicas is confirmed and a new class of materials is brought to the study of the mechanisms of silica pathogenicity.

Effects of yew alkaloids and related compounds on guinea-pig isolated perfused heart and papillary muscle.

The mechanical and electrical effects of selected yew alkaloids were studied on two different cardiac preparations: the isolated coronary perfused heart and the isolated papillary muscle of the guinea-pig. In the isolated heart, the Winterstein acid type alkaloids 1, 2 and 3 induced electrical and mechanical effects similar to those reported after yew intoxication (negative inotropic effect, block of atrio-ventricular conduction), but the coronary flow was unchanged. Taxine B (1), the most potent compound of this group, reduced cardiac contractility and the maximum rate of depolarisation of the action potential in the isolated papillary muscle, acting as a class I antiarrhythmic drug. In the isolated heart, the cinnamates 4 and 5, corresponding to the degradation products of 1 and 3, exerted arrhythmogenic effect due to a reduction of coronary flow. No alterations in electrical and contractile activities were in fact recorded after perfusion of the isolated papillary muscle with 4. Taxine A (6) and the taxane alcohol 7, corresponding to the terpenoid core of 3 had no significant cardiac effect. Our results suggest that the poisonous properties of the yew tree are probably due to the combined activity of alkaloids of the Winterstein acid type and their corresponding cinnamtes, which can reduce both the excitability and the coronary flow of the heart.