Chiara Florio

Nanoparticle dermal absorption and toxicity: a review of the literature

IntroductionNanotechnologies are among the fastest growing areas of scientific research and have important applications in a wide variety of fields. The data suggest that in the future workers and consumers exposed to nanoparticles will significantly increase.Dermal absorption and toxicity of nanoparticlesAt now there are gaps in understanding about the human and environmental risk that manufactured nanoparticles pose for occupational exposed people and for consumers. There is a need for assessing the health and environmental impacts, the nanoparticles life cycle, the human exposure routes, the behavior of nanoparticles in the body, and the risk for workers. Possible routes of entry into the body include inhalation, absorption through the skin or digestive tract, injection, and absorption or implantation for drugs delivery systems. In particular, dermal absorption and skin penetration of nanoparticles needs a better evaluation because few and contradictory data are present in the literature, mainly on titanium dioxide.ConclusionsThere are limited data on carbon-based nanoparticles and very few data on other metal nanoparticles increasingly used in industry. The article reviews the literature on the percutaneous absorption of nanoparticles and their effect on skin.

A protein phosphatase 2A inhibition assay for a fast and sensitive assessment of okadaic acid contamination in mussels

The specific inhibitory activity exerted by okadaic acid on protein phosphatase 2A was used to assess the presence of okadaic acid in mussels, using a commercially available protein phosphatase 2A preparation. Under the conditions used, okadaic acid inhibits the enzymatic activity dose-dependently, with an IC50 = 0.26 ng/ml (0.32 nM). The assay is accurate and reproducible. Okadaic acid was detected in concentrations as low as 0.063 ng/ml in aqueous solutions and 2 ng/g in mussel digestive glands. Thirty naturally contaminated mussel samples were submitted to the protein phosphatase 2A inhibition assay as well as to an ELISA assay and to a MTT cytotoxicity assay, with similar results. The proposed assay is sensitive, rapid and does not require expensive equipment. These characteristics make it a good candidate for employment in the routine assessment of okadaic acid shellfish contamination.

Adenosine A1 receptors modulate anxiety in CD1 mice

Abstract The effect of the selective adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) was investigated in CD1 mice by the elevated plus-maze and the light/dark test, two models for measuring anxiety in rodents. CCPA, administered IP, had an anxiolytic effect at 0.3 nmol/kg in the elevated plus-maze and at 1 nmol/kg in the light/dark test. Brain levels of 22 nM were found after administration of 100 nmol/kg CCPA, as measured by ex vivo binding experiments. These values are consistent with the occupancy of adenosine A1 but not A2 receptors by CCPA, and suggest that the anxiolytic-like action of CCPA may be mediated by centrally located adenosine A1 receptors. Both CPT, a selective adenosine A1 receptor antagonist, and IBMX, a non-selective adenosine antagonist, had an anxiogenic effect in the two tests. It is thus possible that purinergic neurons may be involved in the tonic modulation of affective state in mice.

Suitability of the MTT-based cytotoxicity assay to detect okadaic acid contamination of mussels

The suitability of a cytotoxicity assay based on the MTT colorimetric method has been evaluated for the detection of okadaic acid in mussels. On KB cells, okadaic acid exhibited a dose-dependent cytotoxic effect, the IC50 being inversely related to the exposure time (IC50 = 6.3 ng/ml, 4.0 ng/ml and 1.1 ng/ml after 24, 48 and 72 hr of contact, respectively). Using a contact time of 24 hr, the MTT cytotoxicity assay is suitable for revealing okadaic acid concentrations in mussel samples as low as 50 ng/g of digestive glands, with a sensitivity higher than that of the commercially available kits for enzyme-linked immunosorbent assay (ELISA). In the okadaic acid concentration range from 50 to 1500 ng/g of digestive glands the MTT cytotoxicity assay showed satisfactory accuracy and reproducibility. A high degree of correlation was found between the okadaic acid content of 16 naturally contaminated samples measured by the MTT cytotoxicity assay and by an ELISA.

Yessotoxins: A toxicological overview

Yessotoxins (YTXs) are polyciclic ether compounds produced by phytoplanktonic dinoflagellates and accumulated in filter feeding shellfish. These toxins can be ingested by humans through contaminated seafood consumption. Initially, YTXs were classified as Diarrhetic Shellfish (DS) toxins but the biological activity of these compounds, which lack of diarrheogenic effects, differs from that of diarrheic toxins. Thus, YTXs have been recently classified as a separate group of algal toxins. Yessotoxin (YTX), homoyessotoxin and 45-hydroxy-homoyessotoxin are lethal after intraperitoneal injection to mice but not after single or repeated oral administration. The target organ seems to be the cardiac muscle cells, where these toxins induce light and electron microscopy ultrastructural changes not only after intraperitoneal injection, but also after oral exposure. On the other hand, di-desulfo-yessotoxin affects liver and pancreas, where it induces fatty degeneration. The mechanisms at the basis of the cardiac effects of YTX and homoyessotoxins are still not completely understood. No short term and chronic toxicity data are available as well as pharmacokinetic studies are lacking. Nevertheless, YTX is known to exert different in vitro activities, such as changes of intracellular calcium and cyclic AMP levels, alteration of cytoskeletal and adhesion molecules, caspases activation and opening of the permeability transition pore of mitochondria. This review reports the current knowledge on the in vivo toxicity and in vitro effects of these toxins.

Synthesis, Biological Evaluation, and Three-Dimensional in Silico Pharmacophore Model for σ1 Receptor Ligands Based on a Series of Substituted Benzo[d]oxazol-2(3H)-one Derivatives

Novel benzo[d]oxazol-2(3H)-one derivatives were designed and synthesized, and their affinities against sigma receptors were evaluated. On the basis of 31 compounds, a three-dimensional pharmacophore model for the sigma(1) receptor binding site was developed using the Catalyst 4.9 software package. The best 3D pharmacophore hypothesis, consisting of one positive ionizable, one hydrogen bond acceptor, two hydrophobic aromatic, and one hydrophobic features provided a 3D-QSAR model with a correlation coefficient of 0.89. The best hypothesis was also validated by three independent methods, i.e., the Fisher randomization test included in the CatScramble functionality of Catalyst, the leave-one-out test, and activity prediction of an additional test set. The achieved results will allow researchers to use this 3D pharmacophore model for the design and synthesis of a second generation of high affinity sigma(1) ligands, as well as to discover other lead compounds for this class of receptors.

Silver nanoparticles exert a long-lasting antiproliferative effect on human keratinocyte HaCaT cell line.

For their antibacterial activity, silver nanoparticles (Ag NPs) are largely used in various commercially available products designed to come in direct contact with the skin. In this study we investigated the effects of Ag NPs on skin using the human-derived keratinocyte HaCaT cell line model. Ag NPs caused a concentration- and time-dependent decrease of cell viability, with IC(50) values of 6.8 ± 1.3 μM (MTT assay) and 12 ± 1.2 μM (SRB assay) after 7 days of contact. A 24h treatment, followed by a 6 day recovery period in Ag NPs-free medium, reduced cell viability with almost the same potency (IC(50)s of 15.3 ± 4.6 and 35 ± 20 μM, MTT and SRB assays, respectively). Under these conditions, no evidence of induction of necrotic events (propidium iodide assay) was found. Apocynin, NADPH-oxidase inhibitor, or N(G)-monomethyl-L-argynine, nitric oxide synthase inhibitor, did not prevent NPs-induced reduction of cell viability. TEM analysis of cells exposed to NPs for 24h revealed alteration of nuclear morphology but only a marginal presence of individual NPs inside the cells. These results demonstrate that on HaCaT keratinocytes a relatively short time of contact with Ag NPs causes a long-lasting inhibition of cell growth, not associated with consistent Ag NPs internalization.

Activation of protein kinase C in neutrophil cytoplasts: Localization of protein substrates and possible relationship with stimulus-response coupling

Treatment of enucleated, granule‐free neutrophil cytoplasts with the protein kinase C activator phorbol 12O‐myristate‐13‐acetate (PMA) causes an increased ‐32P‐incorporation into a variety of polypeptides. Permeabilization of PMA‐stimulated, 32P‐labeled cytoplasts by 0.01% digitonin fully releases the majority of these phosphorylated proteins. A statistically significant correlation is found between the extent of PMA‐induced activation of generation of Superoxide anion (O− 2) and the phosphorylation of a cytosolic polypeptide with an apparent M r, of 46000, whose ‐32P‐labeling is also enhanced by the treatment of cytoplasts with 1‐oleyl‐2‐acetylglycerol, the Ca2+ ionophore ionomycin or latex beads. Furthermore, treatment of cytoplasts with the protein kinase C inhibitor trifluoperazine markedly inhibits the 32P‐labeling of proteins in the 40000 M r range, including the 46 kDa polypeptide, and almost totally abolishes the activation of O− 2 production by PMA.

Co-activation of protein kinase C and NADPH oxidase in the plasma membrane of neutrophil cytoplasts.

Enucleated, granule-free neutrophil cytoplasts, which in hypotonic media fully release cytosolic components and generate ghosts, have been used to study the cell localization of protein kinase C (PK-C). Treatment of cytoplasts with phorbol myristate acetate, a potent activator of neutrophil functions, triggers translocation of PK-C from the cytosol to the plasma membrane, with an activity recovery of 83 +/- 16%. In the ghost fraction, PK-C catalyzes the phosphorylation of polypeptides with an apparent mol. wt. of 115K, 89K, 79K, 62K, 47K and 19K. From the plasma membrane PK-C can be extracted in an active form by Triton X-100 but not by EGTA. Translocation of PK-C is already evident at 5 sec and plateaus at about 50 sec. Activation of plasmalemmal, O-2 generating NADPH oxidase by the phorbol ester is delayed by about 20 sec with respect to the activation of PK-C. Dose/response experiments show that the pattern of activation of O-2 generation by cytoplasts strictly superimposes with the pattern of PK-C translocation.

Effects of the marine toxin palytoxin on human skin keratinocytes: Role of ionic imbalance

Palytoxin (PLTX), a marine toxin identified in Palythoa zoanthid corals and Ostreopsis dinoflagellates, represents an increasing hazard for human health. Recently, dermatological problems have been associated to cutaneous exposure to PLTX during Ostreopsis blooms arising the need for experimental data characterizing PLTX effects on the skin. This study highlights in vitro the cytotoxic effects of PLTX on human keratinocytes (HaCaT cell line). A short time exposure (4h) to PLTX reduced mitochondrial activity (MTT assay), cell mass (SRB assay) and plasma membrane integrity (LDH leakage) with different potencies: EC₅₀ values of 6.1 ± 1.3×10⁻¹¹, 4.7 ± 0.9 × 10⁻¹⁰ M and 1.8 ± 0.1 × 10⁻⁸ M, respectively. PLTX effect on mitochondrial activity was ouabain- and Na+-sensitive, but only partially sensitive to removal of Ca²+ ions. One hour exposure to the toxin also induced a Na+-dependent and Ca²+-independent superoxide anion production. These results indicate that among the chain of intracellular events following the interaction of PLTX with the Na+/K+-ATPase the first and crucial step is the increased intracellular Na+ concentration that triggers a sequence of cell dysfunction involving mitochondrial affection and oxidative stress, leading to an irreversible cell death. The PLTX concentrations inducing cytotoxicty seem to be lower than those of potential cutaneous human exposure during Ostreopsis ovata blooms, indicating the harmful potential of the toxin.