Anna Maria Ragnelli

Effects of single and multi walled carbon nanotubes on macrophages: Cyto and genotoxicity and electron microscopy

Production of nanotechnology-based materials is increasing worldwide: it is essential to evaluate their potential toxicity. Among these nanomaterials, carbon nanotubes (CNTs) have tremendous potential in many areas of research and applications. We have investigated the cyto- and genotoxic effects of single and multi-walled CNTs (SWCNTs, MWCNTs) and carbon black (CB) on the mouse macrophage cell line RAW 264.7. Specifically we have investigated inflammatory response, release of tumor necrosis factor-α (TNF-α), intracellular reactive oxygen species (ROS) production, cell death (both necrosis and apoptosis), chromosomal aberrations and cellular ultrastructural alteration caused by CB, MWCNTs and SWCNTs. Our data confirm that both CNTs and CB are cyto and geno-toxic to RAW 264.7 mouse macrophages. CNTs exposure induced ROS release, necrosis and chromosomal aberrations but did not cause an inflammatory response. In addition CNTs induce ultrastructural damage and apoptosis. CNTs penetrate the cell membrane and individual MWCNTs are seen associated with the nuclear envelope.

Peroxisome Proliferator-Activated Receptors (PPARs) and related transcription factors in differentiating astrocyte cultures

Peroxisome proliferator-activated receptors (PPARs), retinoid X receptors (RXRs), CCAAT/enhancer binding proteins (C/EBPs) and beta-catenin are transcription factors involved in cell differentiation. The aim of this work was to investigate the occurrence and variations of these proteins during astrocyte differentiation. Primary cultures of mouse cortical astrocytes were characterized using nestin, A2B5 and glial fibrillary acidic protein (GFAP) as differentiation markers, during a period of 21 days in vitro (DIV). Glycogen and triglyceride accumulation were also studied. At 3 DIV the cultures were mainly constituted by neural progenitor cells, as assessed by their immunofluorescent pattern. At this time PPARs and beta-catenin were localized to the cytoplasm. Interestingly, some cells contained Oil Red O-positive lipid droplets. Between 7 and 21 DIV, nestin decreased, while GFAP increased, indicating ongoing astroglial differentiation. beta-catenin, predominantly nuclear at 7 DIV, later localized to membranes. Redistribution of all three PPAR isotypes from the cytoplasm to the nucleus was observed starting from 7 DIV. Between 7 and 14 DIV, C/EBPalpha, PPARalpha, RXRalpha and glycogen content increased. Between 14 and 21 DIV, PPARbeta/delta decreased, while PPARgamma, C/EBPbeta and delta and lipid droplet-containing cells increased. At 21 DIV both A2B5-/GFAP+ and A2B5+/GFAP+ cells were predominantly observed, indicating differentiation toward type-1 and type-2 astrocytes, although the presence of GFAP- cells demonstrates the persistence of neural precursors in the culture even at this time point. In conclusion, our results, reporting modifications of PPARs, RXRs, C/EBPs and beta-catenin during culture time, strongly suggest the involvement of these transcription factors in astrocyte differentiation. Specifically, beta-catenin translocation from the nucleus to plasma membrane, together with PPARbeta/delta decrease and C/EBPalpha increase, could be related to decreased proliferation at confluence, while PPARalpha and gamma and all C/EBPs could participate in differentiation processes, such as glycogenesis and lipidogenesis.

AGE-DEPENDENT ULTRASTRUCTURAL ALTERATIONS AND BIOCHEMICAL RESPONSE OF RAT SKELETAL MUSCLE AFTER HYPOXIC OR HYPEROXIC TREATMENTS

This work deals with the antioxidant enzymatic response and the ultrastructural aspects of the skeletal muscle of young and aged rats kept under hypoxic or hyperoxic normobaric conditions. It is in fact well known that the supply of oxygen at concentrations higher or lower than those occurring under normal conditions can promote oxidative processes that can cause tissue damage. The enzymes investigated were both those directly involved in reactive oxygen species (ROS) scavenging (superoxide dismutase, catalase and selenium-dependent glutathione peroxidase), and those challenged with the detoxication of cytotoxic compounds produced by the action of ROS on biological molecules (glutathione transferase, glyoxalase I, glutathione reductase), in order to obtain a comparative view of the defence strategies used with respect to aging. Our results support the hypothesis that one of the major contributors to the aging process is the oxidative damage produced at least in part by an impairment of the antioxidant enzymatic system. This makes the aged organism particularly susceptible to oxidative stress injury and to the related degenerative diseases, especially in those tissues with high demand for oxidative metabolism.

Truffle tyrosinase: Properties and activity

Abstract The present paper investigates the l -3,4-dihydroxyphenylalanine oxidase (EC 1.14.18.1) and l -tyrosine 3-monooxygenase (EC 1.14.18.1) activities of truffles of the genus Tuber , a highly pigmented group of Ascomycetes. The laccase (EC 1.10.2.1) activity has also been explored in the homogenate supernatants from these mushrooms. The effects of various inhibitors of tyrosinase, of buffer concentration, temperature and pH on the tyrosinase activity of truffle cytosols have been investigated. The K m values of l -3,4-dihydroxyphenylalanine and l -tyrosine have been calculated and are in the range of those found in other mushrooms (i.e. 0.37 mM and 2.70 mM respectively).The polyacryamide gel electrophoretic pattern of the l -3,4-dihydroxyphenylalanine oxidase activities of different species of truffles have been obtained. Moreover, the truffle pigment formation and localization have been histochemically investigated and correlated with the reproductive differentiation.

Morphofunctional mitochondrial response to methylglyoxal toxicity in Bufo bufo embryos.

Methylglyoxal (2-oxopropanal) is a reactive alpha-oxoaldehyde that can be formed endogenously mainly as a by-product of glycolytic pathway. It is a cytotoxic compound with significant antiproliferative properties as it can bind, under physiological conditions, to nucleic acids and proteins, forming stable adducts. We have recently shown that exogenous methylglyoxal (150-600 microM) is highly toxic for amphibian embryos where it produces, when added to the culture water, inhibition of cell proliferation in the early developmental stages, followed by severe malformations and strongly reduced embryonic viability. In this work we investigate the morphofunctional effect of methylglyoxal on the common toad B. bufo embryo mitochondria in order to verify if its dysmorphogenetic action might be also ascribed to impairment of mitochondrial functions. The mitochondria were isolated from embryos at the developmental stages of morula, neural plate and operculum complete and developing in the presence of 600 microM methylglyoxal. The results show that exogenous methylglyoxal is highly toxic at mitochondrial level, where it produces proliferation, swelling and membrane derangement. As a consequence, mitochondria from treated embryos show decreased oxidative phosphorylation efficiency, as indicated by the significant reduction both of the respiratory control index values and of the embryonic ATP content. On the basis of these data, it is possible that the methylglyoxal-induced embryonic malformations as well as the strongly reduced viability might be also ascribed to energy depletion.

Liposome-entrapped tyrosinase: a tool to investigate the regulation of the Raper-Mason pathway.

The effect of the entrapment of mushroom tyrosinase (EC 1.14.18.1) within liposomes on the enzyme activity and Km vs. L-3,4-dihydroxyphenylalanine is reported in the present work; the effect of cholesterol insertion within liposome membranes on the enzyme activity has also been studied. The oxidation rates of various monophenols and diphenols by free and liposome-integrated mushroom tyrosinase were measured and the oxidation latencies vs. different substrates investigated. The different substrates are apparently oxidized according to the properties of the substituents as electron donors or acceptors; the Km values vs. L-3,4-dihydroxyphenylalanine calculated on measuring O2 consumption are higher than those calculated on measuring the dopachrome production rates. It is interesting that natural substrates of tyrosinase are oxidized according to a negative catalysis by the liposome-entrapped enzyme; this point is discussed in relation to the well known cytotoxicity of some intermediates of the Raper-Mason pathway.

In Vivo Inflammatory Effects of Ceria Nanoparticles on CD-1 Mouse: Evaluation by Hematological, Histological, and TEM Analysis

The attention on CeO2-NPs environmental and in vivo effects is due to their presence in diesel exhaust and in diesel filters that release a more water-soluble form of ceria NPs, as well as to their use for medical applications. In this work, acute and subacute in vivo toxicity assays demonstrate no lethal effect of these NPs. Anyhow, performing in vivo evaluations on CD-1 mouse systems, we demonstrate that it is even not correct to assert that ceria NPs are harmless for living systems as they can induce status of inflammation, revealed by hematological-chemical-clinical assays as well as histological and TEM microscope observations. TEM analysis showed the presence of NPs in alveolar macrophages. Histological evaluation demonstrated the NPs presence in lungs tissues and this can be explained by assuming their ability to go into the blood stream and lately into the organs (generating inflammation).

Tyrosinase expression during black truffle development: From free living mycelium to ripe fruit body

The present work studies the expression of tyrosinase (monophenol:diphenol oxygen oxidoreductase, EC 1.14.18.1) during the development of the black truffle Tuber melanosporum Vittad., an ectomycorrhizal fungus of great biological and economic interest. As widely reported in the literature, melanins and the enzymes that synthesize them, are of paramount importance in fungal development and sexual differentiation. Tyrosinase and laccase are the enzymes that produce melanins from monophenols and diphenols. We have detected tyrosinase expression from the stage of free living mycelium, through the mychorrizal stage and the six fruit body developmental stages by measuring the levels of tyrosinase mRNA by quantitative PCR (q-PCR), spectrophotometry, histochemistry, immunohistochemistry and electrophoresis. Tyrosinase is always expressed, from the free living mycelium to the ripe fruit body developmental stages, when it is very low. The switching off of the tyrosinase gene during T. melanosporum development when the fruit body is ripe and no more cell walls are to be built is discussed in relation of thioflavour production. Specific primers, prepared from the cloned T. melanosporum tyrosinase cDNA were used for the q-PCR and the deduced aminoacid sequences of the CuA and CuB binding sites were compared to those of various ascomycetes and basidiomycetes.

Truffle Development and Interactions with the Biotic Environment

The nature and structure of the substances involved in black truffle morphogenetic development as well as in the interactions with the biotic environment are reviewed. The development of mycelial pellets, which form sporocarp primordia, is marked by a high L-DOPA oxidase and tyrosinase hydroxylase activity originating from a layer of cells just under the outermost one. This layer gives birth to cells which have both an outward and an inward orientation, the external ones gradually grow into isodiametric cells while the internal start branching into the ascus-bearing veins. In peridial cells and spores, tyrosinase activity produces allomelanins (dihydroxynaphtalene or cumaric derivates) which participate in cell wall structure. Some volatile compounds (alcohols and aldehydes with 2–5 C) are produced before spore formation, phenolic and sulphur compounds along with ketones and esters appear only later, when also a steady decrease in tyrosinase activity occurs. Unripe truffle substances are normal fungal metabolites produced via the pyruvate pathway. Some of these seem to be responsible for a strong grass-growing inhibition and a patent modification of the micro-population of the hydnosphere. Soil fungi and the plants tested are affected by three aldheydes (2-methyl propanal, 2-methyl butanal and 3-methyl butanal), the plants also by two alcohols (2-methyl butanol and 3-methyl butanol), even at very low concentrations. In contrast, a strain of truffle Pseudomonas showed a tolerance to much higher concentrations of these substances. While dimethyl sulphide, produced only by ripe truffles, seemed to act as an attractant of spore-spreading animals. By all accounts, truffle sporocarp morphogenesis is initiated by the tyrosinase activity and the subsequent truffle development affects all components of its biotic environment.

Human glioblastoma ADF cells express tyrosinase, L-tyrosine hydroxylase and melanosomes and are sensitive to L-tyrosine and phenylthiourea.

Melanocytes and neuroblasts share the property of transforming L‐tyrosine through two distinct metabolic pathways leading to melanogenesis and catecholamine synthesis, respectively. While tyrosinase (TYR) activity has been shown to be expressed by neuroblastoma it remains to be established as to whether also glioblastomas cells are endowed with this property. We have addressed this issue using the human continuous glioblastoma cell line ADF. We demonstrated that these cells possess tyrosinase as well as L‐tyrosine hydroxylase (TH) activity and synthesize melanosomes. Because the two pathways are potentially cyto‐genotoxic due to production of quinones, semiquinones, and reactive oxygen species (ROS), we have also investigated the expression of the peroxisomal proliferators activated receptor α (PPARα) and nuclear factor‐kB (NFkB) transcription factor as well the effect of L‐tyrosine concentration on cell survival. We report that L‐tyrosine down‐regulates PPARα expression in ADF cells but not neuroblastoma and that this aminoacid and phenylthiourea (PTU) induces apoptosis in glioblastoma and neuroblastoma.