Uliana De Simone

Comparative cellular toxicity of titanium dioxide nanoparticles on human astrocyte and neuronal cells after acute and prolonged exposure.

Although in the last few decades, titanium dioxide nanoparticles (TiO₂NPs) have attracted extensive interest due to their use in wide range of applications, their influences on human health are still quite uncertain and less known. Evidence exists indicating TiO₂NPs ability to enter the brain, thus representing a realistic risk factor for both chronic and accidental exposure with the consequent needs for more detailed investigation on CNS. A rapid and effective in vitro test strategy has been applied to determine the effects of TiO₂NPs anatase isoform, on human glial (D384) and neuronal (SH-SY5Y) cell lines. Toxicity was assessed at different levels: mitochondrial function (by MTT), membrane integrity and cell morphology (by calcein AM/PI staining) after acute exposure (4-24-48 h) at doses from 1.5 to 250 μg/ml as well as growth and cell proliferation (by clonogenic test) after prolonged exposure (7-10 days) at sub-toxic concentrations (from 0.05 to 31 μg/ml). The cytotoxic effects of TiO₂NPs were compared with those caused by TiO₂ bulk counterpart treatment. Acute TiO₂NP exposure produced (i) dose- and time-dependent alterations of the mitochondrial function on D384 and SH-SY5Y cells starting at 31 and 15 μg/ml doses, respectively, after 24h exposure. SH-SY5Y were slightly more sensitive than D384 cells; and (ii) cell membrane damage occurring at 125 μg/ml after 24h exposure in both cerebral cells. Comparatively, the effects of TiO₂ bulk were less pronounced than those induced by nanoparticles in both cerebral cell lines. Prolonged exposure indicated that the proliferative capacity (colony size) was compromised at the extremely low TiO₂NP doses namely 1.5 μg/ml and 0.1 μg/ml for D384 and SH-SY5Y, respectively; cell sensitivity was still higher for SH-SY5Y compared to D384. Colony number decrease (15%) was also evidenced at ≥0.2 μg/ml TiO₂NP dose. Whereas, TiO₂ bulk treatment affected cell morphology only. TiO₂ internalization in SH-SY5Y and D384 cells was appreciated using light microscopy. These findings indicated, that (i) human cerebral SH-SY5Y and D384 cell lines exposed to TiO₂NPs were affected not only after acute but even after prolonged exposure at particularly low doses (≥ 0.1 μg/ml), (ii) these in vitro critical doses were comparable to literature brain Ti levels detected in lab animal intranasally administered with TiO₂NP and associated to neurotoxic effects. In summary, the applied cell-based screening platform seems to provide effective means to initial evaluation of TiO₂NP toxicity on CNS.

Assessment of cellular responses after short- and long-term exposure to silver nanoparticles in human neuroblastoma (SH-SY5Y) and astrocytoma (D384) cells

Silver nanoparticle (AgNP, 20 nm) neurotoxicity was evaluated by an integrated in vitro testing protocol employing human cerebral (SH-SY5Y and D384) cell lines. Cellular response after short-term (4–48 h, 1–100 μg/ml) and prolonged exposure (up to 10 days, 0.5–50 μg/ml) to AgNP was assessed by MTT, calcein-AM/PI, clonogenic tests. Pulmonary A549 cells were employed for data comparison along with silver nitrate as metal ionic form. Short-term data: (i) AgNP produced dose- and time-dependent mitochondrial metabolism changes and cell membrane damage (effects starting at 25 μg/ml after 4 h: EC50s were 40.7 ± 2.0 and 49.5 ± 2.1 μg/ml for SH-SY5Y and D384, respectively). A549 were less vulnerable; (ii) AgNP doses of ≤ 18 μg/ml were noncytotoxic; (iii) AgNO3 induced more pronounced effects compared to AgNP on cerebral cells. Long-term data: (i) low AgNP doses (≤1 μg/ml) compromised proliferative capacity of all cell types (cell sensibility: SHSY5Y > A549 > D384). Colony number decrease in SH-SY5Y and D384 was 50% and 25%, respectively, at 1 μg/ml, and lower dose (0.5 μg/ml) was significantly effective towards SH-SY5Y and pulmonary cells; (ii) cell proliferation activity was more affected by AgNO3 than AgNPs. In summary, AgNP-induced cytotoxic effects after short-term and prolonged exposure (even at low doses) were evidenced regardless of cell model types.

Naturally-occurring porphyrins in a spontaneous-tumour bearing mouse model

An increase in naturally-occurring porphyrins has been described in the blood of subjects bearing different kinds of tumours, that has been proposed as an additional parameter for the diagnosis of occult cancer, although at present the reason for the phenomenon is not exactly defined. In this work the increase of porphyrins in plasma of tumour-bearing subjects has been investigated in parallel with their occurrence in other tissues, considering the systemic iron homeostasis subversion taking place in the presence of cancer. The transgenic female MMTV-neu mouse-developing spontaneous mammary adenocarcinoma has been used as an experimental model, in comparison to non-transgenic C1 mouse as a control. The spleen, accomplishing both hemocatheretic and hemopoietic functions in rodents, and the liver have been considered because of their deep engagement in heme metabolism, entailing both the fate of protoporphyrin IX (PpIX) as its ultimate precursor, and iron homeostasis. Investigations have been performed by means of microspectrofluorometric and image analysis of tissue autofluorescence (AF), and histochemical detection of non-heme iron. In tumour-bearing mouse, along with a marked PpIX presence in tumour, a PpIX enhancement in spleen and liver is observed, that is accompanied by a significant increase in plasma. The phenomenon can be related to a systemic alteration of heme metabolism induced by tumour cells to face their survival and proliferation requirements.

Human liver autofluorescence: an intrinsic tissue parameter discriminating normal and diseased conditions.

Autofluorescence (AF) emission is an intrinsic parameter that can provide real‐time information on morpho‐functional properties of biological tissue, being strictly related with their biochemical composition and structural organization. The diagnostic potentials of AF‐based techniques have been investigated on normal, fibrotic, and steatotic liver tissues, in reference to histological features as evidenced by specific histochemical stainings.

In Vitro Toxicity Evaluation of Engineered Cadmium-Coated Silica Nanoparticles on Human Pulmonary Cells

Cytotoxicity of cadmium-containing silica nanoparticles Cd-SiO2NPs (0.05–100 µg/mL) versus SiO2NPs and CdCl2 was evaluated by an in vitro test battery in A549 by assessing (i) mitochondrial function, (ii) membrane integrity/cell morphology, (iii) cell growth/proliferation, (iv) apoptotic pathway, (v) oxidative stress, after short- (24–48 h) and long-term (10 days) exposure. Both Cd-SiO2NPs and CdCl2 produced dose-dependent cytotoxic effects: (i) MTT-assay: similar cytotoxicity pattern was observed at both 24 and 48 h, with a more Cd-SiO2NPs pronounced effect than CdCl2. Cd-SiO2NPs induced mortality (about 50%) at 1 μg/mL, CdCl2 at 25 μg/mL; (ii) calcein-AM/PI staining: decrease in cell viability, noticeable at 25 μg/mL, enhanced markedly at 50 and 100 μg/mL, after 24 h. Cd-SiO2NPs induced higher mortality than CdCl2 (25% versus 4%, resp., at 25 μg/mL) with further exacerbation after 48h; (iii) clonogenic assay: exposure for longer period (10 days) compromised the A549 proliferative capacity at very low dose (0.05 μg/mL); (iv) a progressive activation of caspase-3 immunolabelling was detected already at 1 μg/mL; (v) GSH intracellular level was modified by all compounds. In summary, in vitro data demonstrated that both Cd-SiO2NPs and CdCl2 affected all investigated endpoints, more markedly after Cd-SiO2NPs, while SiO2NPs influenced GSH only.

Liver autofluorescence properties in animal model under altered nutritional conditions

Autofluorescence spectroscopy is a promising and powerful approach for an in vivo, real time characterization of liver functional properties. In this work, preliminary results on the dependence of liver autofluorescence parameters on the nutritional status are reported, with particular attention to vitamin A and lipid accumulation in liver tissue. Normally fed and 24 h starving rats were used as animal models. Histochemical and autofluorescence analysis showed that lipids and vitamin A colocalize in the liver parenchyma. Fasting condition results in a parallel increase in both lipids and vitamin A. Autofluorescence imaging and microspectrofluorometric analysis carried out on unfixed, unstained tissue sections under 366 nm excitation, evidenced differences in both spectral shape and response to continuous irradiation between liver biopsies from fed and starving rats. As to photobleaching, in particular, fitting analysis evidenced a reduction of about 85% of the signal attributable solely to vitamin A during the first 10 s of irradiation. The tissue whole emission measured in fed and starving rat livers exhibited reductions of about 35% and 52%, respectively, that are closely related to vitamin A contents. The findings open interesting perspectives for the set up of an in situ, real time diagnostic procedure for the assessment of liver lipid accumulation, exploiting the photophysical properties of vitamin A.

Cytotoxicity and proliferative capacity impairment induced on human brain cell cultures after short- and long-term exposure to magnetite nanoparticles.

Since magnetic iron oxide nanoparticles (IONP) as magnetite (Fe3O4NPs) have potential applications in life sciences, industrial fields and biomedical care, the risks for occupational, general population and patients rises correspondingly. Excessive IONP accumulation in central nervous system (CNS) cells can lead to a disruption of normal iron metabolism/homeostasis, which is a characteristic hallmark resembling that of several neurodegenerative disorders. Fe3O4NPs‐ versus Fe3O4 bulk‐induced toxic effects have been assessed in two human CNS cells namely astrocytes (D384) and neurons (SH‐SY5Y) after short‐term exposure (4–24‐48 h) to 1–100 μg ml−1, and long‐term exposure to lower concentrations. Short‐term Fe3O4NPs induced significant concentration‐ and time‐dependent alterations of mitochondrial function in D384 (25–75% cell viability decrease): effects started at 25 μg ml−1 after 4 h, and 1 μg ml−1 after 48 h. SH‐SY5Y were less susceptible: cytotoxicity occurred after 48 h only with 35–45% mortality (10–100 μg ml−1). Accordingly, a more marked intracellular iron accumulation was observed in astrocytes than neurons. Membrane integrity was unaltered in both CNS cell types. Lowering Fe3O4NP concentrations (0.05–10 μg ml−1) and prolonging the exposure time (up to 10 days), D384 toxicity was again observed (colony number decrease at ≥0.05 μg ml−1, morphology alterations and colony size reduction at ≥0.5 μg ml−1). Effects on SH‐SY5Y appeared at the highest concentration only. Fe3O4 bulk was always remarkably toxic toward both cells. In summary, human cultured astrocytes were susceptible to both Fe3O4NP and bulk forms following short‐term and extended exposure to low concentrations, while neurons were more resistant to NPs. Cellular iron overload may trigger adverse responses by releasing iron ions (particularly in astrocytes) thus compromising the normal functions of CNS. Copyright

Human co-culture model of neurons and astrocytes to test acute cytotoxicity of neurotoxic compounds

Alternative methods and their use in planning and conducting toxicology experiments have become essential for modern toxicologists, thus reducing or replacing living animals. Although in vitro human co-culture models allow the establishment of biologically relevant cell–cell interactions that recapitulate the tissue microenvironment and better mimic its physiology, the number of publications is limited specifically addressing this scientific area and utilizing this test method which could provide an additional valuable model in toxicological studies. In the present study, an in vitro model based on central nervous system (CNS) cell co-cultures was implemented using a transwell system combining human neuronal cells (SH-SY5Y cell line) and glial cells, namely astrocytes (D384 cell line), to investigate neuroprotection of D384 on SH-SY5Y and vice versa. The model was applied to test acute (24-48 hours) cytotoxicity of 3 different neurotoxicants: (1) methyl mercury (1-2.5 μM), (2) Fe3O4 nanoparticles (1-100 μg/mL), and (3) methylglyoxal (0.5-1 mM). Data were compared to mono-cultures evaluating the mitochondrial function and cell morphology. The results clearly showed that all compounds tested affected the mitochondrial activity and cell morphology in both mono-culture and co-culture conditions. However, astrocytes, when cultured together with neurons, diminish the neurotoxicant-induced cytotoxic effects that occurred in neurons cultured alone, and astrocytes become more resistant in the presence of neurons. This human CNS co-culture system seems a suitable cell model to feed high-throughput acute screening platforms and to evaluate both human neuronal and astrocytic toxicity and neuroprotective effects of new and emerging materials (eg, nanomaterials) and new products with improved sensitivity due to the functional neuron–astrocyte metabolic interactions.

Enhanced toxicity of silver nanoparticles in transgenic Caenorhabditis elegans expressing amyloidogenic proteins.

Abstract The increasing number of applications of silver nanoparticles (AgNP) prompted us to assess their toxicity in vivo. We have investigated their effects on wild type and transgenic Caenorhabditis elegans (C. elegans) strains expressing two prototypic amyloidogenic proteins: β2-microglobulin and Aβ peptide3–42. The use of C. elegans allowed us to highlight AgNP toxicity in the early phase of the worm’s life cycle (LC50 survival, 0.9 µg/ml). A comparative analysis of LC50 values revealed that our nematode strains were more sensitive to assess AgNP toxicity than the cell lines, classically used in toxicity tests. Movement and superoxide production in the adult population were significantly affected by exposure to AgNP; the transgenic strains were more affected than the wild type worms. Our screening approach could be applied to other types of nanomaterials that can enter the body and express any nanostructure-related bioactivities. We propose that C. elegans reproducing the molecular events associated with protein misfolding diseases, e.g. Alzheimer’s disease and systemic amyloidosis, may help to investigate the specific toxicity of a range of potentially harmful molecules. Our study suggests that transgenic C. elegans may be used to predict the effect of chemicals in a “fragile population”, where an underlying pathologic state may amplify their toxicity.

Human neuronal cell based assay: A new in vitro model for toxicity evaluation of ciguatoxin

Ciguatoxins (CTXs) are emerging marine neurotoxins representing the main cause of ciguatera fish poisoning, an intoxication syndrome which configures a health emergency and constitutes an evolving issue constantly changing due to new vectors and derivatives of CTXs, as well as their presence in new non-endemic areas. The study applied the neuroblastoma cell model of human origin (SH-SY5Y) to evaluate species-specific mechanistic information on CTX toxicity. Metabolic functionality, cell morphology, cytosolic Ca2+i responses, neuronal cell growth and proliferation were assessed after short- (4-24h) and long-term exposure (10days) to P-CTX-3C. In SH-SY5Y, P-CTX-3C displayed a powerful cytotoxicity requiring the presence of both Veratridine and Ouabain. SH-SY5Y were very sensitive to Ouabain: 10 and 0.25nM appeared the optimal concentrations, for short- and long-term toxicity studies, respectively, to be used in co-incubation with Veratridine (25μM), simulating the physiological and pathological endogenous Ouabain levels in humans. P-CTX-3C cytotoxic effect, on human neurons co-incubated with OV (Ouabain+Veratridine) mix, was expressed starting from 100pM after short- and 25pM after long-term exposure. Notably, P-CTX-3C alone at 25nM induced cytotoxicity after 24h and prolonged exposure. This human brain-derived cell line appears a suitable cell-based-model to evaluate cytotoxicity of CTX present in marine food contaminated at low toxic levels and to characterize the toxicological profile of other/new congeners.