Claudia Petrarca

Palladium nanoparticles induce disturbances in cell cycle entry and progression of peripheral blood mononuclear cells: paramount role of ions.

There is concern about the possible toxicity of palladium nanoparticles (Pd-NP), as they are released in the environment through many applications. We previously studied the toxicity of Pd-NP at high concentrations; here we address the possible toxicity of Pd-NP at low, subtoxic doses. In particular, we have exposed normal human PBMC entering into the first in vitro mitotic division to Pd-NP and to Pd(IV) ions to evaluate ROS generation and cell cycle progression. We have measured a statistically significant increase of intracellular ROS in Pd(IV) exposed cells, but not in Pd-NP exposed cells. TEM revealed accumulation of lipid droplets and autophagic and mitophagic vacuoles, which appeared more conspicuous in cells exposed to Pd(IV) ions than to Pd-NP. Pd-NP were visible in the cytoplasm of Pd-NP exposed cells. Pd-NP addition was associated with a significant increase of cells within the G0/G1-phase and a significant reduction in GS- and G2/M-phases. Cells exposed to Pd(IV) ions showed a significant amplification of these cell cycle alterations. These results suggest that ions, per se or released by NPs, are the true inducers of Pd toxicity. It will be essential to verify whether the observed disturbance represents a temporary response or might result in permanent alterations.

Cobalt nano-particles modulate cytokine in vitro release by human mononuclear cells mimicking autoimmune disease

: The use of particles from micro to nanoscale provides benefits to diverse scientific fields, but because a large percentage of their atoms lie on the surface, nanomaterials could be highly reactive and pose potential risks to humans. Due to their wide range of application, Cobalt nano-particles are of great interest both in industry and in life-science. To date, there are few studies on Co nano-particle toxicology. In this respect, this study aims at evaluating in vitro the potential interference of Co nano-particles on the production of several cytokines (IL-2, IL-4, IL-6, IL-10, IFNgamma and TNFalpha) by PBMCs, comparing their effects to those of Co micro-particles and Co solution (CoCl2). Cells were cultured in Opticell flasks with escalating concentrations (10-5, 10-6 and 10-7 M), of Co nano and micro-particles and CoCl2 or without metal. Cytokines were quantified in the supernatants using a human Th1/Th2 cytokine cytometric bead array. Co micro-particles showed a greater inhibitory effect compared to other Co forms. Its inhibitory activity was detected at all concentrations and towards all cytokines, whereas Co solutions selectively inhibited IL-2, IL-10 and TNF-alpha at maximal concentration. Co nano-particles induced an increase of TNF-alpha and IFN-gamma release and an inhibition of IL-10 and IL-2: a cytokine pattern similar to that detected in the experimental and clinical autoimmunity. On the basis of the obtained data, immune endpoints should be sought in the next series of studies both in vitro and in vivo in subjects exposed to cobalt nano-particles.The mainstay of therapy for patients with advanced prostate cancer still remains androgen deprivation, although response to this is invariably temporary. Most of the patients develop hormone-refractory disease resulting in progressive clinical deterioration and, ultimately, death. Until recently there has been no standard chemotherapeutic approach for hormone refractory prostate cancer (HRPC), the major benefits of chemotherapy being only palliative. The studies combining mitoxantrone plus a corticosteroid demonstrated that chemotherapy could be given to men with symptomatic HRPC with minimal toxicity and a significant palliation could be provided. Recently, results from 2 phase III randomized clinical trials demonstrating that a combination of docetaxel plus prednisone can improve survival in men with HRPC have propelled docetaxel-based therapy into the forefront of treatment options for these patients as the new standard of care. There is a promising activity of new drug combinations such as taxanes plus vinca alkaloids; bisphosphonates are assuming a prominent role in prostate therapy through their ability to prevent skeletal morbidity. Combinations of classic chemotherapeutic agents and biological drugs began to be tested in phase II-III trials and the first results appear interesting. This article focuses on combinations recently evaluated or under clinical development for the treatment of HRPC.

Autophagy as an ultrastructural marker of heavy metal toxicity in human cord blood hematopoietic stem cells

Stem cells are a key target of environmental toxicants, but little is known about their toxicological responses. We aimed at developing an in-vitro model based on adult human stem cells to identify biomarkers of heavy metal exposure. To this end we investigated the responses of human CD34+ hematopoietic progenitor cells to hexavalent chromium (Cr[VI]) and cadmium (Cd). Parallel cultures of CD34+ cells isolated from umbilical cord blood were exposed for 48 h to 0.1 microM and 10 microM Cr(VI) or Cd. Cultures treated with 10 microM Cr(VI) or Cd showed marked cell loss. Ultrastructural analysis of surviving cells revealed prominent autophagosomes/autophagolysosomes, which is diagnostic of autophagy, associated with mitochondrial damage and replication, dilatation of the rough endoplasmic reticulum and Golgi complex, cytoplasmic lipid droplets and chromatin condensation. Treated cells did not show the morphologic hallmarks of apoptosis. Treatment with 0.1 microM Cr(VI) or Cd did not result in cell loss, but at the ultrastructural level cells showed dilated endoplasmic reticulum and evidence of mitochondrial damage. We conclude that autophagy is implicated in the response of human hematopoietic stem cells to toxic concentrations of Cr(VI) and Cd. Autophagy, which mediates cell survival and death under stress, deserves further evaluation to be established as biomarker of metal exposure.

Inhibitory effect of quercetin on tryptase and interleukin-6 release, and histidine decarboxylase mRNA transcription by human mast cell-1 cell line.

Mast cells are involved in inflammatory processes and in allergic reactions where immunologic stimulation leads to degranulation and generation of numerous cytokines and inflammatory mediators. Mast cells have been proposed as an immune gate to the brain, as well as sensors of environmental and emotional stress, and are likely involved in neuropathologic processes such as multiple sclerosis. Among mast cell products, the protease tryptase could be associated with neurodegenerative processes through the activation of specific receptors (PARs) expressed in the brain, while interleukin (IL)-6 likely causes neurodegeneration and exacerbates dysfunction induced by other cytokines; or it could have a protective effect against demyelinisation. In this report we show that quercetin, a natural compound able to act as an inhibitor of mast cell secretion, causes a decrease in the release of tryptase and IL-6 and the down-regulation of histidine decarboxylase (HDC) mRNA from human mast cell (HMC)-1 cells. As quercetin dramatically inhibits mast cell tryptase and IL-6 release and HDC mRNA transcription by HMC-1 cell line, these results nominate quercetin as a therapeutical compound in association with other therapeutical molecules for neurological diseases mediated by mast cell degranulation.

Engineered metal based nanoparticles and innate immunity

Almost all people in developed countries are exposed to metal nanoparticles (MeNPs) that are used in a large number of applications including medical (for diagnostic and therapeutic purposes). Once inside the body, absorbed by inhalation, contact, ingestion and injection, MeNPs can translocate to tissues and, as any foreign substance, are likely to encounter the innate immunity system that represent a non-specific first line of defense against potential threats to the host. In this review, we will discuss the possible effects of MeNPs on various components of the innate immunity (both specific cells and barriers). Most important is that there are no reports of immune diseases induced by MeNPs exposure: we are operating in a safe area. However, in vitro assays show that MeNPs have some effects on innate immunity, the main being toxicity (both cyto- and genotoxicity) and interference with the activity of various cells through modification of membrane receptors, gene expression and cytokine production. Such effects can have both negative and positive relevant impacts on humans. On the one hand, people exposed to high levels of MeNPs, as workers of industries producing or applying MeNPs, should be monitored for possible health effects. On the other hand, understanding the modality of the effects on immune responses is essential to develop medical applications for MeNPs. Indeed, those MeNPs that are able to stimulate immune cells could be used to develop of new vaccines, promote immunity against tumors and suppress autoimmunity.

Cytotoxicity and morphological transforming potential of cobalt nanoparticles, microparticles and ions in Balb/3T3 mouse fibroblasts: an in vitro model

Abstract We previously described the behaviour of different cobalt forms, i.e., cobalt nanoparticles (CoNP), cobalt microparticles (CoMP) and cobalt ions (Co2+), in culture medium (dissolution, interaction with medium components, bioavailability) as well as their uptake and intracellular distribution in Balb/3T3 mouse fibroblasts (Sabbioni, Nanotoxicology, 2012). Here, we assess the cytotoxicity and morphological transformation of CoNP compared not only to Co2+, but also to CoMP and to released Co products. Cytotoxicity reached maximum at 4-h exposure, with ranking CoMP > CoNP > Co2+. However, if we consider toxicity as a function of intracellular Co, toxicity of the ionic forms seems to prevail over the particles. Co forms other than Co2+ released from particles had toxicity intermediate between particles and ions. Alterations in concentrations of essential elements (Cu, Mg, Zn) in cells exposed to Co particles may contribute to toxicity. Both CoMP and CoNP (but not Co2+ and other released Co forms) induced morphological transformation (CoMP > CoNP). This was dependent on reactive oxygen species production and lipid peroxidation, as indicated by inhibition of type III foci with ascorbic acid. The present results suggest that the previously demonstrated massive mitochondrial and nuclear Co internalisation and DNA adduct formation by CoMP and CoNP (Sabbioni, Nanotoxicology, 2012) induce toxicity and transformation. On the contrary, the role of ions released by particles in culture medium is negligible. Thus, both the chemical and the physical properties of Co particles contribute to cytotoxicity and morphological transformation.

Interaction with culture medium components, cellular uptake and intracellular distribution of cobalt nanoparticles, microparticles and ions in Balb/3T3 mouse fibroblasts

Abstract The mechanistic understanding of nanotoxicity requires the physico-chemical characterisation of nanoparticles (NP), and their comparative investigation relative to the corresponding ions and microparticles (MP). Following this approach, the authors studied the dissolution, interaction with medium components, bioavailability in culture medium, uptake and intracellular distribution of radiolabelled Co forms (CoNP, CoMP and Co2+) in Balb/3T3 mouse fibroblasts. Co2+ first saturates the binding sites of molecules in the extracellular milieu (e.g., albumin and histidine) and on the cell surface. Only after saturation, Co2+ is actively uptaken. CoNP, instead, are predicted to be internalised by endocytosis. Dissolution of Co particles allows the formation of Co compounds (CoNP-rel), whose mechanism of cellular internalisation is unknown. Co uptake (ranking CoMP > CoNP > Co2+) reached maximum at 4 h. Once inside the cell, CoNP spread into the cytosol and organelles. Consequently, massive amounts of Co ions and CoNP-rel can reach subcellular compartments normally unexposed to Co2+. This could explain the fact that the nuclear and mitochondrial Co concentrations resulted significantly higher than those obtained with Co2+.

Immunotoxicity and Sensitizing Capacity of Metal Compounds Depend on Speciation

Immunotoxicity of metal compounds is an issue of great importance due to the recent industrial application of metals with unknown toxicity on the immune system and the discovery of metal intermediary compounds not sufficiently studied yet. In this report we show results of our study on the immunotoxicity of the following metals: the Platinum group elements (Platinum, Palladium, Rhodium), Titanium and Arsenic. We applied functional and non functional assays and investigated both innate and adaptive immune systems, in particular, cell proliferation, cytokine production by PBMCs and O−2 production by neutrophils. We obtained the following results: only some Ti compounds (Titanocene, Ti ascorbate and Ti oxalate) show immunotoxicity. Trivalent As compounds (Sodium arsenite and tetraphenyl arsonium chloride) are more immunotoxic than the other investigated As compounds. Genotoxicity of Pt group compounds is in the following order: Pt < Rh < Pd. Immunotoxicity of Pt group compounds is in the following order: Pd < Pt < Rh. Lymphocytes and macrophages show a different reaction of neutrophils to metal toxicity. We can conclude that these studies show that metal immunotoxicity depends on speciation. In general speciation provides additional and often essential information in evaluating metal toxicity. However, there are many difficulties in applying speciation in investigating toxico-kinetic aspects to many metals, mainly due to the lack of information about the existence and significance of species and to the lack of analytical methods for measuring species in biological samples.

Autophagy in hematopoietic stem/progenitor cells exposed to heavy metals: Biological implications and toxicological relevance.

The inherent toxicity of many metal compounds, together with their widespread environmental distribution, raises concerns of potential health hazards. Little is known about the impact of these important environmental toxicants on adult stem/progenitor cells, necessary for tissue homeostasis and repair. We recently reported that autophagy is implicated in the response of hematopoietic stem/progenitor cells to toxic concentrations of hexavalent chromium (Cr[VI]) and cadmium (Cd), two well known carcinogenic heavy metal cations. Autophagy may lead to cell death if carried out too extensively, but also acts as a survival pathway in cells under stress. In stem/progenitor cells, an autophagic phenotype could mitigate metal-induced toxicity, contributing to the conservation of tissue renewal capability. Given the key role of toxic damage to adult stem/progenitor cells in cancer, it is necessary to investigate whether autophagic responses modulate the carcinogenic potential of exposure to heavy metals during stem/progenitor cell differentiation. Addendum to: Di Gioacchino M, Petrarca C, Perrone A, Farina M, Sabbioni E, Hartung T, Martino S, Esposito DL, Lotti LV, Mariani-Costantini R. Autophagy as an ultrastructural marker of heavy metal toxicity in human cord blood hematopoietic stem cells. Sci Total Environ. 2008; 392:50-8.

The proinflammatory interleukin-21 elicits anti-tumor response and mediates autoimmunity.

Interleukins (IL) are inflammatory proteins (except IL-4 and IL-10) that modulate the immune system (1-4). IL-2l induced inflammation in vivo, based on recruitment of neutrophil and monocyte populations (5-8). This cytokine is similar in primary sequence and structure to IL-2 and IL-15 and is a member of the type I cytokine superfamily which includes IL-2, IL-4, IL-7, IL-9, IL-ll, IL-12, IL-13, IL-15 and Colony Stimulating Factors (CSFs). IL-2l is a multifunctional cytokine having roles in both innate and adaptive immune responses of the Th 1 type (9-11) with an effect on proliferation, apoptosis and differentiation of T cells, NK cells, dendritic cells and B cells (12-15) and lymphocytes function (16-18) (Fig. 1). IL-21, like other members of this family, is a gamma-chain dependent cytokine playing a prominent role in promoting and maintaining T cell populations (19-21). Human and murine IL-2l are 57% identical at the amino acid level, display a 4helix-bundle-type with homology to IL-2, IL-4 and IL-15 and have a unique receptor, IL-21R. The exon and intron structure of the IL-2 and IL-2l genes are very similar and are related to each other. IL21R is a type I cytokine receptor that acts through the interaction with the common gamma chain and is expressed in lymphoid tissues: thymus, spleen, peripheral blood leukocytes such as T cells (CD4+ and CD8+), B cells, NK cells, dendritic cells and several cell lines (22-24). IL-2l R possesses alpha, beta and gamma chain heterotrimer. The beta and gamma chain are essential for signal transduction while the alpha subunit seems to be more involved in high-affinity binding conversion. An anti-gamma chain antibody inhibits cell proliferation induced by IL-2l, suggesting that the gamma chain plays an essential role in IL-2l signal transduction (2527). The generation of IL-21R-deficient mice with normal lymphoid cells revealed dysfunction in immunoglobulin IgG1 production and an increase in IgE responses in immunized animals (28-30). IL-2l needs the common gamma chain to mediate the intracellular survival and/or mitogenic signalling, which occurs through the essential transducermolecule JAK3 (5, 31); however, three major pathways are involved in IL-2l signalling ( JAKISTAT, MAPK and PI3K) to promote and maintain T lymphocyte populations (32-34). Survival and differentiation of B cells is mediated by IL-2l through down-regulation of anti-apoptotic proteins and up-regulation of proapoptotic proteins (35-38).