Carla Falugi

Apoptosis as a specific biomarker of diazinon toxicity in NTera2-D1 cells.

The NTera2/D1 (NT2) cell line, which was derived from a human teratocarcinoma, exhibits properties that are characteristics of a committed neuronal precursor at an early stage of differentiation. Its property to express a whole set of molecules related to the cholinergic neurotransmission system, including active acetylcholinesterase (AChE, EC 3.1.1.7) makes it a good alternative model for testing the effects of neurotoxic compounds, such as organophosphorus (OP) insecticides, whose primary target is the inhibition of AChE activity. Recent findings have elucidated the role of AChE in the modulation of apoptosis, but the mechanisms are still rather obscure. NT2 cells exposed to the OP insecticide diazinon at concentrations ranging between 10(-4) and 10(-5)M showed a time-dependent enhancement of cell death. When exposed at 10(-6)M diazinon showed higher cell viability than control samples up to 72 h, followed by a decreasing phase. The cell death caused by the exposures showed a number of features characteristic of apoptosis, including membrane and mitochondrial potential changes. We suggest the hypothesis that such behaviour is due to a dynamic balance between activated and blocked acetylcholine receptors that in turn trigger electrical events and caspase cascade.

α7-Nicotinic Acetylcholine Receptors Affect Growth Regulation of Human Mesothelioma Cells Role of Mitogen-Activated Protein Kinase Pathway

This study presents data suggesting that both human mesothelioma (cell lines and human mesothelioma biopsies) and human normal mesothelial cells express receptors for acetylcholine and that stimulation of these receptors by nicotine prompted cell growth via activation of nicotinic cholinergic receptors. Thus, these data demonstrate that: (a) human mesothelioma cells and human biopsies of mesothelioma as well as of normal pleural mesothelial cells express functionally α-7 nicotinic acethlycholine receptors, evaluated by α-bungarotoxin-FITC binding, receptor binding assay, Western blot, and reverse transcription-PCR; (b) choline acetyltransferase immunostaining is present in mesothelioma cells; (c) mesothelioma cell growth is modulated by the cholinergic system in which agonists (i.e., nicotine) has a proliferative effect, and antagonists (i.e., curare) has an inhibitory effect, evaluated by cell cloning, DNA synthesis and cell cycle; (d) nicotine induces Ca+2 influx, evaluated by [45Ca2+] uptake, and consequently activation of mitogen-activated protein kinase pathway (extracellular signal-regulated kinase and p90RSK phosphorylation), evaluated by Western blot; and (e) apoptosis mechanisms in mesothelioma cells are under the control of the cholinergic system (nicotine antiapoptotic via induction of nuclear factor-κB complexes and phosphorylation of Bad at Ser112; curare proapoptotic via G0-G1 arrest p21waf-1 dependent but p53 independent). The involvement of the nonneuronal cholinergic system in mesothelioma appears reasonable and open up new therapeutic strategies.

Toxicity of metal oxide nanoparticles in immune cells of the sea urchin

The potential toxicity of stannum dioxide (SnO₂), cerium dioxide (CeO₂) and iron oxide (Fe₃O₄) nanoparticles (NPs) in the marine environment was investigated using the sea urchin, Paracentrotus lividus, as an in vivo model. We found that 5 days after force-feeding of NPs in aqueous solutions, the three NPs presented different toxicity degrees, depending on the considered biomarkers. We examined: 1) the presence of the NPs in the coelomic fluid and the uptake into the immune cells (coelomocytes); 2) the cholinesterase activity and the expression of the stress-related proteins HSC70 and GRP78; 3) the morphological changes affecting cellular compartments, such as the endoplasmic reticulum (ER) and lysosomes. By Environmental Scanning Electron Microscope (ESEM) analysis, coupled with Energy Dispersive X-ray Spectroscopy (EDS) we found that NPs were uptaken inside coelomocytes. The cholinesterases activity, a well known marker of blood intoxication in vertebrates, was greatly reduced in specimens exposed to NPs. We found that levels of stress proteins were down-regulated, matching the observed ER and lysosomes morphological alterations. In conclusion, this is the first study which utilizes the sea urchin as a model organism for biomonitoring the biological impact of NPs and supports the efficacy of the selected biomarkers.

High surface adsorption properties of carbon-based nanomaterials are responsible for mortality, swimming inhibition, and biochemical responses in Artemia salina larvae

We investigated the effects of three different carbon-based nanomaterials on brine shrimp (Artemia salina) larvae. The larvae were exposed to different concentrations of carbon black, graphene oxide, and multiwall carbon nanotubes for 48 h, and observed using phase contrast and scanning electron microscopy. Acute (mortality) and behavioural (swimming speed alteration) responses and cholinesterase, glutathione-S-transferase and catalase enzyme activities were evaluated. These nanomaterials were ingested and concentrated in the gut, and attached onto the body surface of the A. salina larvae. This attachment was responsible for concentration-dependent inhibition of larval swimming, and partly for alterations in the enzyme activities, that differed according to the type of tested nanomaterials. No lethal effects were observed up to 0.5mg/mL carbon black and 0.1mg/mL multiwall carbon nanotubes, while graphene oxide showed a threshold whereby it had no effects at 0.6 mg/mL, and more than 90% mortality at 0.7 mg/mL. Risk quotients calculated on the basis of predicted environmental concentrations indicate that carbon black and multiwall carbon nanotubes currently do not pose a serious risk to the marine environment, however if uncontrolled release of nanomaterials continues, this scenario can rapidly change.

Developmental abnormalities and changes in cholinesterase activity in sea urchin embryos and larvae from sperm exposed to engineered nanoparticles

The objective of this study is to examine the toxicity of engineered nanoparticles (NPs) that are dispersed in sea water by using an in vivo model. Because many products of nanotechnology contain NPs and are commonly used and well-established in the market, the accidental release of NPs into the air and water is quite possible. Indeed, at the end of their life cycle, some NPs are inevitably released into waste water and can reach marine ecosystem and affect the organisms there. Although there are few data on the presence of NPs in the marine environment, our awareness of their potential impact on environmental and organismal health is growing. Shallow-water benthonic organisms such as sea urchins provide planktonic larvae as a trophic base for finfish juveniles and are exposed to water from estuaries and precipitation. Such organisms can therefore be directly affected by NPs that are dispersed into those media. We evaluated the effects of exposure to different concentrations of nanosilver, titanium oxide and cobalt NPs on the sperm of the sea urchin Paracentrotus lividus by analyzing the functionality and the morphology and biochemistry of the first developmental stages of the sea urchin. Sperm were exposed to sea water containing suspensions of NPs ranging from 0.0001 mg/L to 1 mg/L. Fertilization ability was not affected, but developmental anomalies were identified in embryos from the gastrula to pluteus stages, including morphological alterations of the skeletal rods. In addition, the enzymatic activity (cholinesterase, ChE) of the larvae was measured. Acetylcholinesterase (AChE) and propionylcholinesterase activity (PrChE) was affected in all of the exposed samples. The results did not vary consistently with the concentration of NP, but controls were significantly different from exposed samples. Exposure of sea urchin to these NPs may cause neurotoxic damage, and the altered ChE activity may be involved in skeletogenic aberrations. In conclusion, the sea urchin represents a suitable and sensitive model for testing the toxicity and effects of engineered NPs that are dispersed in sea water.

RPR-115135, a farnesyltransferase inhibitor, increases 5-FU- cytotoxicity in ten human colon cancer cell lines: Role of p53

A new non peptidic farnesyltransferase inhibitor, RPR‐115135, in combination with 5‐FU was studied in 10 human colon cancer cell lines (HCT‐116, RKO, DLD‐1, Colo‐320, LoVo, SW‐620, HT‐29, HCT‐15, Colo‐205 and KM‐12) carrying several mutations but well characterized for p53 and Ras status. We found that there was a slight tendency (not statistically significant) for the p53 inactivated cells to be less sensitive to 5‐FU after 6 days continuous treatment. Simultaneous administration of RPR‐115135 and 5‐FU, at subtoxic concentrations, resulted in a synergistic enhancement of 5‐FU cytotoxicity in the p53 wildtype cells (HCT‐116, RKO, DLD‐1, Colo‐320, LoVo). In the p53 mutated cells (SW‐620, HT‐29, HCT‐15, Colo‐205, KM‐12) the effect was very complicated. In HCT‐15 the combination resulted in antagonism, in KM‐12 in antagonism or in synergy (at different concentrations) and in SW‐620, HT‐29 and Colo‐205 cells in synergy but only when 5‐FU was administered at high concentrations. Growth inhibition could be accounted for on the basis of a specific cell cycle arrest phenotype (G2‐M arrest), as assayed by flow cytometry, only in the p53 functioning cell lines. The combination RPR‐115135 + 5‐FU increases apoptotic events only in these cell lines. In the mutated cell lines no major alterations on cell cycle arrest phenotype and no induction of apoptosis was observed. Although RPR‐115135 can potentiate the effect of 5‐FU in cells in which p53 function is disrupted, these data suggest strongly that RPR‐115135 significantly enhances the efficacy of 5‐FU only when p53 is functioning.

Biological effects of a neurotoxic pesticide at low concentrations on sea urchin early development. a terathogenic assay

Dose-dependent terathogenic effects of an organophosphate insecticide were found during early sea urchin development. This biological assay is low cost, easy to measure, and allows to detect the effects of the exposure of organisms to the active principle at concentrations lower than the acceptable daily intake (ADI) for man. Effects were found independently from the stage of exposure, and were major as earlier exposure occurred. The stronger effects were exerted on the elongation of the skeletal rods, that was easily measured by following the migration of primary mesenchyme cells, labelled by WGA (wheat germ agglutinin).

Toxicity and transfer of metal oxide nanoparticles from microalgae to sea urchin larvae

Nanoparticles (NPs) contained in commercial products are released and enter into the aquatic ecosystem, posing serious possible risks to the environment and affecting the food chain. Therefore, investigating the potential toxicity of NPs on aquatic organisms has become an important issue. This study assessed the toxicity and trophic transfer of metal oxide NPs from marine microalgae (Cricosphaera elongata) to the larvae of the sea urchin Paracentrotus lividus. Larvae (24 h old) were fed on 2000 cell mL−1 48 h of microalgae contaminated with 5 mg L−1 of several metal oxide NPs (SiO2, SnO2, CeO2, Fe3O4) for 15 days. Larval viability and development were monitored from the 4-arm stage to the 8-arm pluteus stage. A significant decrease in survival was observed in larvae fed with microalgae exposed to SiO2 and CeO2 NPs. Abnormal development, characterised by skeletal degeneration and altered rudiment growth, was observed in all larvae fed with contaminated NP algae. Our findings revealed that SiO2 and CeO2 NPs exerted a toxic effect in the trophic interaction analysed, by reducing sea urchin larval viability, and all metal oxide NPs induced toxicological effects. In conclusion, metal oxide NPs may enter the food chain and become bioavailable for marine organisms, affecting their development.

Role of the Non-Neuronal Human Cholinergic System in Lung Cancer and Mesothelioma: Possibility of New Therapeutic Strategies

Acetylcholine (Ach), one of the most important examples of a neurotransmitter, represents a phylogenetically old molecule, widely distributed from bacteria to humans. The finding that neuronal Ach receptors (nAChRs) are present in non-neuronal cells raised some interesting issues related to their specific activity. In humans, different studies have showed that many lung cancer cells expressed nAchRs and that low concentrations of nicotine blocked the induction of apoptosis in these cells. A recent study presents data that SCLC express a cholinergic autocrine loop that can regulate cell growth. Such work demonstrates that SCLC cells have a cholinergic phenotype and that ACh exerts as an autocrine growth factor in human lung tumors. Recently it has been shown that human malignant pleural mesothelioma express a cholinergic system, involved in cell growth regulation. Hence, mesothelioma cell growth as well as normal mesothelial cells growth is modulated by the cholinergic system in which agonists (i.e. nicotine) has a proliferative effect and antagonists (i.e. curare) has an inhibitory effect. Furthermore apoptosis mechanisms in mesothelioma cells are under the control of the cholinergic system (nicotine antiapoptotic via induction of NF-kappaB complexes and phosphorilation of Bad at Serine(112), curare proapoptotic via G(0)-G(1) arrest p21(waf-1)-dependent, but p53-independent). The involvement of the non-neuronal cholinergic system in lung cancer and mesothelioma appears reasonable and open up new therapeutic strategies.

Pre-adipocytes commitment to neurogenesis 1: Preliminary localisation of cholinergic molecules

A great effort has recently been made to obtain human stem cells able to differentiate into cholinergic neurons, as a number of diseases are associated to the cholinergic neuron loss, degeneration or incorrect function (Alzheimers disease and motor neuron disease). A stem cell population (i.e. pre‐adipocytes) is present in the adipose stromal compartment. Pre‐adipocytes, like the mesodermic derivative cells, retain high plasticity and potentiality to convert in vitro from one phenotype into many others, and they can be isolated from adult adipose tissue. Pre‐adipocytes committed in vitro to neural differentiation were followed up to the acquisition of neural morphology. Acetylcholinesterase and choline acetyltransferase are expressed from the native cell stage, with different localisations and roles during neural commitment. Western blots show the beginning of a new synthesis of these enzymes at 4 weeks of culture of neurogenic pre‐adipocytes, in parallel with neural morphology. The passage of the choline‐acetyltransferase immunoreactivity from cytoplasmic to membrane localisation shows the possible onset of catalytic activity and the histochemical reaction confirms the activity of acetylcholinesterase. This explains the possibility of obtaining cholinergic‐like phenotype from pre‐adipocytes.