Evaluation of the cytotoxic, genotoxic and inflammatory effects induced by the exposure to mineral fibres in in vitro human cellular models

Exposure to mineral fibres represents a serious occupational and environmental hazard, since it leads to chronic lung inflammation with the subsequent development of pneumoconiosis, fibrotic pulmonary diseases, lung cancer and malignant mesothelioma. However, since the toxicity and carcinogenicity of mineral fibres is profoundly interwoven with a broad variety of crystal-chemical-physical characteristics, the biogeochemical mechanisms through which mineral fibres, and especially asbestos, induce adverse effects in vivo are yet to be completely understood. The first part of this study aimed at investigating the different possible toxicity mechanisms of UICC standard crocidolite, chrysotile from Balangero and erionite from Jersey, which are respectively representative of the classes of amphibole asbestos, serpentine asbestos and fibrous erionite. The acute damaging effects exerted by these mineral fibres during the first 24 h of exposure were evaluated by performing a comparative study in an in vitro THP-1 cellular model of M0, M1 and M2 macrophages. The toxicity mechanisms of the three mineral fibres appeared to differ significantly. Crocidolite seemed to exert its toxic effects mostly as a result of its biodurability, ROS production, cytokine release and DNA damage. Chrysotile, due to its low biodurability, displayed toxic effects correlated with the release of toxic metals and the production of ROS and cytokines. Other mechanisms were involved in explaining the toxicity of biodurable fibrous erionite, which induced lower ROS and toxic metal release, but exhibited a cation exchange capacity able to alter the intracellular homeostasis of important cations. Interestingly, M2 macrophages, which are characteristically known for countering the inflammatory response, in the presence of asbestos fibres and erionite, exacerbated this process by secreting pro-inflammatory mediators. For the second part of this project, representative batches of short (length < 5 µm) and long (length > 5 µm) chrysotile fibres were prepared by cryogenic milling of a commercial chrysotile extracted from an open pit mine near Yasny (Russia). To gain new insights into the toxicity mechanisms of these size-separated fractions of chrysotile, their cytotoxic and genotoxic activity was investigated in an in vitro cellular model based on human THP-1-derived M0 macrophages and HECV endothelial cells, both separately as well as in a co-culture setup. At all time-points, both chrysotile fractions displayed significant acute cytotoxic effects, with results that were comparable to the well-known damaging effects of crocidolite. In particular, the long fraction (> 5 μm) of chrysotile showed a notably higher cytotoxic potential, causing ROS production, DNA damage and the transcriptional upregulation of inflammatory mediators in M0 macrophages. On the other hand, the short fraction of chrysotile displayed a significant degree of cytotoxicity and genotoxicity in vitro, which is quite intriguing considering the ongoing debate regarding the potential toxicity of short asbestos fibres (< 5 μm). It should also be noted that in HECV cells the mineral fibres caused toxicity mainly as a result of direct cell membrane damage, whereas in M0 macrophages the apoptotic mechanism had a fundamental role in the induction of cell death

Synchrotron radiation methods for studying biological interaction of fibres: an overview

In the last decade, synchrotron-based techniques have emerged as effective tools for investigating biological systems at the sub-cellular level. In asbestos-related studies, X-ray microscopy, combined with X-ray Fluorescence (XRF) and X-ray absorption near edge structure (XANES) enable successfully investigating the interaction between fibres and cells. Microscopy and chemical imaging provide very detailed maps, thus chemically and morphologically characterizing the biological interaction of fibres while monitoring different toxicity mechanisms. Additionally, the combination of microchemical and structural information can clarify metal mobilization processes, highlighting both their intracellular spatial distribution and the possible changes in their valence state induced by the fibres-cells interaction. The activities within the PRIN 2017-FIBRES project included the utilization of multidisciplinary techniques and expertise to study the interaction of different fibres (chrysotile, erionite and crocidolite) with THP-1-derived macrophages. The numerous experiments carried out at TwinMic (Elettra synchrotron, Trieste, Italy) and ID21 (ESRF, Grenoble, France) beamlines - here discussed and generally presented - offered the opportunity to review the invaluable contribution of synchrotron-based techniques in studying mineral fibres and their biological interaction to step forward in the understanding of asbestos toxicity and carcinogenicity

Effect of Crosslinking Type on the Physical-Chemical Properties and Biocompatibility of Chitosan-Based Electrospun Membranes

Chitosan nanofibrous membranes are prepared via an electrospinning technique and explored as potential wound healing patches. In particular, the effect of a physical or chemical crosslinking treatment on the mat morphological, mechanical, water-related, and biological properties is deeply evaluated. The use of phosphate ions (i.e., physical crosslinking) allows us to obtain smooth and highly homogenous nanofibers with an average size of 190 nm, whereas the use of ethylene glycol diglycidyl ether (i.e., chemical crosslinking) leads to rougher, partially coalesced, and bigger nanofibers with an average dimension of 270 nm. Additionally, the physically crosslinked mats show enhanced mechanical performances, as well as greater water vapour permeability and hydrophilicity, with respect to the chemically crosslinked ones. Above all, cell adhesion and cytotoxicity experiments demonstrate that the use of phosphate ions as crosslinkers significantly improves the capability of chitosan mats to promote cell viability owing to their higher biocompatibility. Moreover, tuneable drug delivery properties are achieved for the physically crosslinked mats by a simple post-processing impregnation methodology, thereby indicating the possibility to enrich the prepared membranes with unique features. The results prove that the proposed approach may lead to the preparation of cheap, biocompatible, and efficient chitosan-based nanofibers for biomedical and pharmaceutical applications

Elicited ROS Scavenging Activity, Photoprotective, and Wound-Healing Properties of Collagen-Derived Peptides from the Marine Sponge <i>Chondrosia reniformis</i>

Recently, the bioactive properties of marine collagen and marine collagen hydrolysates have been demonstrated. Although there is some literature assessing the general chemical features and biocompatibility of collagen extracts from marine sponges, no data are available on the biological effects of sponge collagen hydrolysates for biomedical and/or cosmetic purposes. Here, we studied the in vitro toxicity, antioxidant, wound-healing, and photoprotective properties of four HPLC-purified fractions of trypsin-digested collagen extracts&#8212;marine collagen hydrolysates (MCHs)&#8212;from the marine sponge C. reniformis. The results showed that the four MCHs have no degree of toxicity on the cell lines analyzed; conversely, they were able to stimulate cell growth. They showed a significant antioxidant activity both in cell-free assays as well as in H2O2 or quartz-stimulated macrophages, going from 23% to 60% of reactive oxygen species (ROS) scavenging activity for the four MCHs. Finally, an in vitro wound-healing test was performed with fibroblasts and keratinocytes, and the survival of both cells was evaluated after UV radiation. In both experiments, MCHs showed significant results, increasing the proliferation speed and protecting from UV-induced cell death. Overall, these data open the way to the use of C. reniformis MCHs in drug and cosmetic formulations for damaged or photoaged skin repair

The Remarkable Antioxidant and Anti-Inflammatory Potential of the Extracts of the Brown Alga Cystoseira amentacea var. stricta

Inflammation and oxidative stress are part of the complex biological responses of body tissues to harmful stimuli. In recent years, due to the increased understanding that oxidative stress is implicated in several diseases, pharmaceutical industries have invested in the research and development of new antioxidant compounds, especially from marine environment sources. Marine seaweeds have shown the presence of many bioactive secondary metabolites, with great potentialities from both the nutraceutical and the biomedical point of view. In this study, 50%-ethanolic and DMSO extracts from the species C. amentacea var. stricta were obtained for the first time from seaweeds collected in the Ligurian Sea (north-western Mediterranean). The bioactive properties of these extracts were then investigated, in terms of quantification of specific antioxidant activities by relevant ROS scavenging spectrophotometric tests, and of anti-inflammatory properties in LPS-stimulated macrophages by evaluation of inhibition of inflammatory cytokines and mediators. The data obtained in this study demonstrate a strong anti-inflammatory effect of both C. amentacea extracts (DMSO and ethanolic). The extracts showed a very low grade of toxicity on RAW 264.7 macrophages and L929 fibroblasts and a plethora of antioxidant and anti-inflammatory effects that were for the first time thoroughly investigated. The two extracts were able to scavenge OH and NO radicals (OH EC50 between 392 and 454 &mu;g/mL; NO EC50 between 546 and 1293 &mu;g/mL), to partially rescue H2O2-induced RAW 264.7 macrophages cell death, to abate intracellular ROS production in H2O2-stimulated macrophages and fibroblasts and to strongly inhibit LPS-induced inflammatory mediators, such as NO production and IL-1&alpha;, IL-6, cyclooxygenase-2 and inducible NO synthase gene expression in RAW 264.7 macrophages. These results pave the way, for the future use of C. amentacea metabolites, as an example, as antioxidant food additives in antiaging formulations as well as in cosmetic lenitive lotions for inflamed and/or damaged skin

Effect of sodium alginate molecular structure on electrospun membrane cell adhesion

Alginate-based electrospun nanofibers prepared via electrospinning technique represent a class of materials with promising applications in the biomedical and pharmaceutical industries. However, to date, the effect of alginate molecular mass and block composition on the biological response of such systems remains to some extent unclear. As such, in the present work, three alginates (i.e., M.pyr, L.hyp, A.nod) with different molecular features are employed to prepare nanofibers whose ability to promote cell adhesion is explored by using both skin and bone cell lines. Initially, a preliminary investigation of the raw materials is carried out via rheological and zeta-potential measurements to determine the different grade of polyelectrolyte behaviour of the alginate samples. Specifically, both the molecular mass and block composition are found to be important factors affecting the alginate response, with long chains and a predominance of guluronic moieties leading to a marked polyelectrolyte nature (i.e., lower dependence of the solution viscosity upon the polymer concentration). Subsequently, physically crosslinked alginate nanofibrous mats are first morphologically characterized via both scanning electron and atomic force microscopy, which show a homogenous and defect-free structure, and their biological response is then evaluated. Noticeably, fibroblast and keratinocyte cell lines do not show significant differences in terms of cell adhesion on the three mats (i.e., 30\u201340% and 10\u201320% with respect to the seeded cells, respectively), with the formers presenting a greater affinity toward the alginate-based nanofibers. Conversely, both the investigated osteoblast cells are characterized by a distinct behaviour depending on the alginate type. Specifically, polysaccharide samples with an evident polyelectrolyte nature are found to better promote cell viability (i.e., cell adhesion in the range 15\u201336% with respect to seeded cells) compared to the ones displaying a nearly neutral behaviour (i.e., cell adhesion in the range 5\u201325% with respect to seeded cells). Therefore, the obtained results, despite being preliminary, suggest that the alginate type (i.e., molecular structure properties) may play a topical role in conditioning the efficiency of healing patches for bone reparation, but it has a negligible effect in the case of skin regeneration

Chitosan–Collagen Electrospun Nanofibers Loaded with Curcumin as Wound-Healing Patches

Composite chitosan-collagen nanofibrous mats embedded with curcumin were prepared via a single-step electrospinning procedure and explored as wound-healing patches with superior biological activity. A mild crosslinking protocol consisting of a short exposure to ammonia vapor and UV radiation was developed to ensure proper stability in physiological-like conditions without affecting the intrinsic biocompatibility of chitosan and collagen. The fabricated composite patches displayed a highly porous, homogeneous nanostructure consisting of fibers with an average diameter of 200 nm, thermal stability up to 200 °C, mechanical features able to ensure protection and support to the new tissues, and water-related properties in the ideal range to allow exudate removal and gas exchange. The release kinetic studies carried out in a simulated physiological environment demonstrated that curcumin release was sustained for 72 h when the mats are crosslinked hence providing prolonged bioactivity reflected by the displayed antioxidant properties. Remarkably, combining chitosan and collagen not only ensures prolonged stability and optimal physical-chemical properties but also allows for better-promoting cell adhesion and proliferation and enhanced anti-bacteriostatic capabilities with the addition of curcumin, owing to its beneficial anti-inflammatory effect, ameliorating the attachment and survival/proliferation rates of keratinocytes and fibroblasts to the fabricated patches

Identification, Purification and Molecular Characterization of Chondrosin, a New Protein with Anti-tumoral Activity from the Marine Sponge Chondrosia Reniformis Nardo 1847

: Chondrosia reniformis is a common marine demosponge showing many peculiarities, lacking silica spicules and with a body entirely formed by a dense collagenous matrix. In this paper, we have described the identification of a new cytotoxic protein (chondrosin) with selective activity against specific tumor cell lines, from C. reniformis, collected from the Liguria Sea. Chondrosin was extracted and purified using a salting out approach and molecular weight size exclusion chromatography. The cytotoxic fractions were then characterized by two-dimensional gel electrophoresis and mass spectrometry analysis and matched the results with C. reniformis transcriptome database. The procedure allowed for identifying a full-length cDNA encoding for a 199-amino acids (aa) polypeptide, with a signal peptide of 21 amino acids. The mature protein has a theoretical molecular weight of 19611.12 and an IP of 5.11. Cell toxicity assays showed a selective action against some tumor cell lines (RAW 264.7 murine leukemia cells in particular). Cell death was determined by extracellular calcium intake, followed by cytoplasmic reactive oxygen species overproduction. The in silico modelling of chondrosin showed a high structural homology with the N-terminal region of the ryanodine receptor/channel and a short identity with defensin. The results are discussed suggesting a possible specific interaction of chondrosin with the Cav 1.3 ion voltage calcium channel expressed on the target cell membranes