Paolo Lucchesi

Genotoxicity of amorphous silica particles with different structure and dimension in human and murine cell lines

Although amorphous silica is used in food products, cosmetics and paints and as vector for drug delivery, data on its potential health hazard are limited. The aim of this study was to investigate the cytotoxic and genotoxic potential of silica particles of different sizes (250 and 500nm) and structures (dense and mesoporous). Dense silica (DS) spheres were prepared by sol-gel synthesis, mesoporous silica particles (MCM-41) were prepared using hexadecyltrimethyl ammonium bromide as a structure-directing agent and tetraethylorthosilicate as silica source. Particles were accurately characterised by dynamic light scattering, nitrogen adsorption, X-ray diffraction and field emission scanning electron microscopy. Murine macrophages (RAW264.7) and human epithelial lung (A549) cell lines were selected for investigation. Genotoxicity was evaluated by Comet assay and micronucleus test. Cytotoxicity was tested by the trypan blue method. Cells were treated with 0, 5, 10, 20, 40 and 80 µg/cm(2) of different silica powders for 4 and 24 h. The intracellular localisation of silica was investigated by transmission electron microscopy. Amorphous particles penetrated into the cells, being compartmentalised within endocytic vacuoles. DS and MCM-41 particles induced cytotoxic and genotoxic effects in A549 and RAW264.7 although to different extent in the two cell lines. A549 were resistant in terms of cell viability, but showed a generalised induction of DNA strand breaks. RAW264.7 were susceptible to amorphous silica exposure, exhibiting both cytotoxic and genotoxic responses as DNA strand breaks and chromosomal alterations. The cytotoxic response of RAW264.7 was particularly relevant after MCM-41 exposure. The genotoxicity of amorphous silica highlights the need for a proper assessment of its potential hazard for human health.

Imogolite: an aluminosilicate nanotube endowed with low cytotoxicity and genotoxicity.

High-aspect-ratio nanomaterials (HARN) (typically, single-walled carbon nanotubes (SWCNT) or multiwalled carbon nanotubes (MWCNT)) impair airway barrier function and are toxic to macrophages. Here, we assess the biological effects of nanotubes of imogolite (INT), a hydrated alumino-silicate [(OH)3Al2O3SiOH] occurring as single-walled NT, on murine macrophages and human airway epithelial cells. Cell viability was assessed with resazurin. RT-PCR was used to study the expression of Nos2 and Arg1, markers of classical or alternative macrophage activation, respectively, and nitrite concentration in the medium was determined to assess NO production. Epithelial barrier integrity was evaluated from the trans-epithelial electrical resistance (TEER). Potential genotoxicity of INT was assessed with comet and cytokinesis-block micronucleus cytome assays. Compared to MWCNT and SWCNT, INT caused much smaller effects on RAW264.7 and MH-S macrophage viability. The incubation of macrophages with INT at doses as high as 120 μg/cm(2) for 72 h did not alter either Nos2 or Arg1 expression nor did it increase NO production, whereas IL6 was induced in RAW264.7 cells but not in MH-S cells. INT did not show any genotoxic effect in RAW264.7 and A549 cells except for a decrease in DNA integrity observed in epithelial A549 cells after treatment with the highest dose (80 μg/cm(2)). No significant change in permeability was recorded in Calu-3 epithelial cell monolayers exposed to INT, whereas comparable doses of both SWCNT and MWCNT lowered TEER. Thus, in spite of their fibrous nature, INT appear not to be markedly toxic for in vitro models of lung-blood barrier cells.

Oocyte growth, follicle cell differentiation and vitellin processing in the stick insect, Carausius morosus Br. (Phasmatodea)

Abstract Ovarian growth in stick insects (Phasmatodea) was examined ultrastructurally and cytochemically with a view to studying: (1) the kinetics of oocyte growth and the staging characteristics of ovarian follicles undergoing vitellogenesis; (2) the endocytic capability of the growing oocyte, including the post-endocytic fate of the vitellins sequestered by the oocyte during vitellogenesis; (3) the differentiation of the follicular epithelium in relation to the appearance of intercellular spaces and the extracellular release of a follicle cell product. These structural observations were interpreted in relation to the nature and kinetics of the vitellin processing in follicles undergoing vitellogenesis.

Ultrastructure of the female gonad of two temnocephalids (Platyhelminthes, Rhabdocoela)

The female gonad of Temnocephala dendyi and T. minor consists of a single germarium and two rows of vitellaria. It is enveloped by an extracellular lamina and accessory cells. Accessory cells are only peripherally located in the germarium while their cytoplasmic projections also fill the spaces between vitellocytes in the vitellarium. The main feature of oocyte maturation is the appearance of chromatoid bodies and the development of rough endoplasmic reticulum (R.E.R.) and Golgi complexes which appear to be correlated with the production of double-structured egg granules. The egg granules, which are localized in the cortical cytoplasm of mature oocytes, contain glycoproteins, are devoid of polyphenols and are similar in structure and composition to the cortical granules observed in some Digenea and Monogenea. Vitellocytes are typical secretory cells with well-developed R.E.R. and Golgi complexes which are involved in the production of shell globules and yolk. The multigranular pattern and the polyphenolic composition of the shell globules of the temnocephalids investigated are similar to those observed in other rhabdocoels, and in some Prolecithophora and Neodermata. This feature may represent a synapomorphy shared by these taxa.

The ultrastructure of the germarium in some Rhabdocoela

Ultrastructural features of the germarium (ovary) have been investigated in several species of Rhabdocoela. The gonad is usually unpaired, small and pear-shaped; it is enveloped by an extracellular lamina and contains oocytes at different stages of maturation. Elongate accessory cells surround germ cells in the peripheral zone of the gonad and can also fill the internal spaces between oocytes with their long, flattened processes. The main features observed during oocyte maturation were the appearance of chromatoid bodies, annulate lamellae, lipid droplets and glycogen particles, and the development of R.E.R. and Golgi complexes which appeared correlated with the production of small inclusions that became localized in the cortical ooplasm of mature germ cells. The inclusions exhibited a different structure in different taxa and contained variable amounts of polyphenols. Cortical inclusions of rhabdocoels most probably represent residual shell-granules and may participate in the capsule-shell formation.

Integument ultrastructure of Oestrus ovis (L.) (Diptera: Oestridae) larvae: Host immune response to various cuticular components

The nasal bot fly, Oestrus ovis, was investigated to establish which specific cuticular component is most immunogenic to infested sheep and how larval cuticle attains a protective role, if any, against the host immune system. To accomplish these goals, larval cuticle was extracted by a variety of agents and tested against immune sera from infested sheep and experimentally immunized rabbits. The cuticle substructure remaining after extraction was examined to localize various immunogenic components. O. ovis larval integument comprises an inner cellular layer, the epidermis, and an overlying cuticle layer. In 3rd instar larvae, the cuticle comprises 2 additional layers: the procuticle with numerous pore canals and the epicuticle which includes the wax canals. Three additional layers, altogether comprising the cuticulin layer, are present external to the epicuticle. The epicuticle is completed by apposition of an amorphous electrondense material extending for up to 1 micron in thickness. When fixed with ruthenium red, cuticle becomes heavily stained all along the epicuticular surface in larvae of all developmental stages. However, in 3rd instar larvae, ruthenium red deposits are restricted to the cuticulin layer alone. By gel electrophoresis, 3rd instar larval cuticle is shown to contain a number of polypeptides ranging in molecular weight from 180 to 4.5 kDa. The number and relative concentration of low molecular weight polypeptides was shown to vary in relation to the extraction media employed. Cuticular fragments examined after extraction exhibit an altered ultrastructure. When tested by immunoblotting, the cuticular polypeptides most reactive against sheep antisera are in the range of 180-56 kDa. A similar reaction was also detected with sera from rabbits infested experimentally with O. ovis larvae. Results are interpreted in relation to differential polypeptide distribution within the larval cuticle and to accessibility of the host immune system.

The female gonad in two species of Microplana (Platyhelminthes, Tricladida, Rhynchodemidae): Ultrastructural and cytochemical investigations

The female gonad of the land planarians Microplana scharffi and Microplana terrestris consists of two small germaria located ventrally in the anterior third of the body and of two ventro‐lateral rows of oblong vitelline follicles distributed between the intestinal pouches. Both these structures are enveloped by a tunica composed of an outer extracellular lamina and an inner sheath of accessory cells. Oocyte maturation is characterized by the appearance of chromatoid bodies and the development of endoplasmic reticulum and Golgi complexes. These organelles appear to be correlated with the production of egg granules with a fenestrated/granular content of medium electron density, about 4–5 μm in diameter, which remain dispersed in the ooplasm of mature oocytes. On the basis of cytochemical tests showing their glycoprotein composition, and their localization in mature oocytes, these egg granules have been interpreted as yolk. In the vitelline follicles, vitellocytes show the typical features of secretory cells with well‐developed rough endoplasmic reticulum and Golgi complexes involved in the production of eggshell globules and yolk. The eggshell globules, which appear to arise from repeated coalescences of two types of Golgi‐derived vesicles, contain polyphenols and, when completely mature, they measure about 1–1,2 μm in diameter and show a meandering/concentric content pattern as is typical of the situation observed in most Proseriata and Tricladida. Mature vitellocytes also contain a large amount of glycogen and lipids as further reserve material. On the basis of the ultrastructural features of the female gonad and in relation to the current literature the two species of rhynchodemids investigated appear to be closely related to the freshwater planarians belonging to the family Dugesiidae. J. Morphol. 2009.

The ultrastructure of the germanium in some Rhabdocoela

Ultrastructure features of the germanium (ovary) have been investigated in several species of Rhabdocoela. The gonad is usually unpaired, small and pear-shaped; it is enveloped by an extracellular lamina and contains oocytes at different stages of maturation. Elongate accessory cells surround germ cells in the peripheral zone of the gonad and can also fill the internal spaces between oocytes with their long, flattened processes. The main features observed during oocyte maturation were the appearance of chromatoid bodies, annulate lamellae, lipid droplets and glycogen particles, and the development of R.E.R. and Golgi complexes which appeared correlated with the production of small inclusions that became localized in the cortical ooplasm of mature germ cells. The inclusions exhibited a different structure in different taxa and contained variable amounts of polyphenols. Cortical inclusions of rhabdocoels most probably represent residual shell-granules and may participate in the capsule-shell formation.

Cysteine proprotease colocalizes with vitellogenin in compound granules of the cockroach fat body

Abstract. A cysteine proprotease has been identified in developing embryos of the cockroach Blattella germanica and found to be a maternally encoded gene product that is transferred endocytically to the oocyte. The present study aims at establishing how this maternally derived proprotease is synthesized, packaged, and secreted during vitellogenesis. To this end, proprotease was localized immunocytochemically in the fat body of postmating females and its localization compared with that of vitellogenin over the same developmental periods. Fat bodies in cockroaches are comprised of two different cell types: trophocytes and bacteriocytes. Data show that proprotease and vitellogenin come to colocalize in compound granules of the fat body trophocytes. While synthesis of vitellogenin can be traced back to granules resulting from the coalescence of Golgi-derived vesicles in the trophocyte cytoplasm, proprotease appears to be localized predominantly on the cytolysosomes of both trophocytes and bacteriocytes. When probed with an anti-proprotease antiserum, bacteria are also positively labeled, regardless of whether they are segregated inside the cytolysosomes or free in the bacteriocyte cytoplasm. Since vitellogenin and proprotease colocalize within the same cell organelle, it is assumed that Golgi-derived vesicles, which contain vitellogenin, may fuse with cytolysosomes bearing proprotease to yield compound secretory granules. To account for the present observations, the origin and role of proprotease are discussed in relation to the turnover of bacteria in the fat body and to the requirements of endosymbiosis.

The female gonad of the epizoic platyhelminth Troglocaridicola sp. (Rhabdocoela, Temnocephalida, Scutariellidae): ultrastructural and cytochemical investigations

The cytoarchitecture of the female gonad of the scutariellid Troglocaridicola sp. has been investigated by means of electron microscopy and cytochemical techniques. It consists of a single germarium and two rows of vitelline follicles, both enveloped by an outer extracellular lamina (EL) and an inner cellular tunica constituted by accessory cells. Some ultrastructural features which differ from the basic pattern of all the other Rhabdocoela studied so far have been found. In the germarium the following are observed: (a) The presence in the oocytes of peripheral translucent vesicles containing glycoproteins, which differ in diameter and substructure from the peripheral egg granules observed in all the other neoophoran Platyhelminthes. These vesicles can be considered an autapomorphic feature of the taxon Troglocaridicola; (b) The presence of proteinaceous acorn-shaped granules which remain scattered in the ooplasm throughout oogenesis and can be interpreted as residual yolk. This situation is shared with some Proseriata and Tricladida; (c) The precence of accessory cell processes between growing and mature oocytes, as is typical of some Proseriata and Tricladida. In the vitellarium, the presence of polyphenolic shell globules whose substructure does not correspond either to the multigranular pattern prevailing in representatives of Eulecithophora (Prolecithophora+Rhabdocoela) or to the homogeneous pattern found in Lecithoepitheliata. They have a meandering/concentric pattern of the content similar to that described in some Proseriata and Tricladida. On the basis of the ultrastructural characteristics of the female gonad described above, the position of Troglocaridicola in the taxon Rhabdocoela is discussed.