Massimiliano G. Bianchi

Non-functionalized multi-walled carbon nanotubes alter the paracellular permeability of human airway epithelial cells

Little information is available upon the effects of carbon nanotubes (CNT) on the airway barrier. Here we study the barrier function of Calu-3 human airway epithelial cells, grown on permeable filters, after the exposure to commercial single-walled or multi-walled CNT, produced through chemical vapour deposition. To assess changes in the paracellular permeability of CNT-treated Calu-3 monolayers, we have measured the trans-epithelial electrical resistance (TEER) and the permeability to mannitol. Multi-walled CNT caused a large decrease in TEER and an increase in mannitol permeability but no substantial alteration in monolayer viability. Single-walled CNT produced much smaller changes of TEER while CNT, synthesized through the arc discharge method, and Carbon Black nanoparticles had no effect. If commercial multi-walled CNT were added during the formation of the tight monolayer, no further increase in trans-epithelial resistance was observed. Moreover, the same nanomaterials, but neither single-walled counterparts nor Carbon Black, prevented the TEER recovery observed after the discontinuation of interleukin-4, a Th2 cytokine that causes a reversible barrier dysfunction in airway epithelia. These findings suggest that commercial multi-walled CNT interfere with the formation and the maintenance of tight junctional complexes in airway epithelial cells.

C6 GLIOMA CELLS DIFFERENTIATED BY RETINOIC ACID OVEREXPRESS THE GLUTAMATE TRANSPORTER EXCITATORY AMINO ACID CARRIER 1 (EAAC1)

The transport of excitatory amino acids (EAA) in CNS is performed by a family of high affinity, sodium dependent carriers. One of these transporters, excitatory amino acid carrier 1 (EAAC1), is known to be regulated by several mechanisms that modify carrier abundance on the plasma membrane. Much less is known on EAAC1 regulation at the level of gene expression. Here we report that, in C6 rat glioma cells, a line recently described to contain neural stem-like cells, EAAC1 is markedly induced by all trans-retinoic acid (ATRA), a well known differentiating agent. Consistently, ATRA stimulates EAA transport, with the maximal effect observed at concentrations>or=1 microM. After 4 days of treatment with 10 microM ATRA, the transport Vmax is fivefold enhanced, Slc1a1 mRNA is increased by 400% compared with control, EAAC1 carrier is sixfold overexpressed and the C6 culture is greatly enriched of cells with bipolar morphology strongly positive for EAAC1 immunoreactivity. Compared with untreated cells, ATRA-treated C6 cells express less Slc1a3 mRNA, for the transporter GLAST, but significantly higher levels of Slc1a2 mRNA, for the transporter GLT-1, although no expression of either protein is detected with Western blot in both untreated and ATRA-treated cells. Consistently, the inhibition pattern of aspartate transport and its stimulation by phorbol esters are indicative of a transport process due to EAAC1 operation. Under the conditions adopted, ATRA treatment causes the induction of proteolipid protein, an oligodendrocytic marker. These results indicate that, in C6 cells, ATRA stimulates the expression of EAAC1, possibly as a step toward oligodendrocytic differentiation, and constitute the first demonstration of the induction of this transporter by a differentiating agent.

Changes in the expression of the glutamate transporter EAAT3/EAAC1 in health and disease.

Excitatory amino acid transporters (EAATs) are high-affinity Na+-dependent carriers of major importance in maintaining glutamate homeostasis in the central nervous system. EAAT3, the human counterpart of the rodent excitatory amino acid carrier 1 (EAAC1), is encoded by the SLC1A1 gene. EAAT3/EAAC1 is ubiquitously expressed in the brain, mostly in neurons but also in other cell types, such as oligodendrocyte precursors. While most of the glutamate released in the synapses is taken up by the “glial-type” EAATs, EAAT2 (GLT-1 in rodents) and EAAT1 (GLAST), the functional role of EAAT3/EAAC1 is related to the subtle regulation of glutamatergic transmission. Moreover, because it can also transport cysteine, EAAT3/EAAC1 is believed to be important for the synthesis of intracellular glutathione and subsequent protection from oxidative stress. In contrast to other EAATs, EAAT3/EAAC1 is mostly intracellular, and several mechanisms have been described for the rapid regulation of the membrane trafficking of the transporter. Moreover, the carrier interacts with several proteins, and this interaction modulates transport activity. Much less is known about the slow regulatory mechanisms acting on the expression of the transporter, although several recent reports have identified changes in EAAT3/EAAC1 protein level and activity related to modulation of its expression at the gene level. Moreover, EAAT3/EAAC1 expression is altered in pathological conditions, such as hypoxia/ischemia, multiple sclerosis, schizophrenia, and epilepsy. This review summarizes these results and provides an overall picture of changes in EAAT3/EAAC1 expression in health and disease.

Proinflammatory Effects of Pyrogenic and Precipitated Amorphous Silica Nanoparticles in Innate Immunity Cells

Amorphous silica nanoparticles (ASNP) can be synthetized via several processes, 2 of which are the thermal route (to yield pyrogenic silica) and the wet route from a solution containing silicate salts (to obtain precipitated, colloidal, mesoporous silica, or silica gel). Both methods of synthesis lead to ASNP that are applied as food additive (E551). Current food regulation does not require that production methods of additives are indicated on the product label, and, thus, the ASNP are listed without mentioning the production method. Recent results indicate, however, that pyrogenic ASNP are more cytotoxic than ASNP synthesized through the wet route. The present study was aimed at clarifying if 2 representative preparations of ASNP, NM-203 (pyrogenic) and NM-200 (precipitated), of comparable size, specific surface area, surface charge, and hydrodynamic radius in complete growth medium, had different effects on 2 murine macrophage cell lines (MH-S and RAW264.7 cells). Our results show that, when incubated in protein-rich fluids, NM-203 adsorbed on their surface more proteins than NM-200 and, once incubated with macrophages, elicited a greater oxidative stress, assessed from Hmox1 induction and ROS production. Flow cytometry and helium ion microscopy indicated that pyrogenic NM-203 interacted with macrophages more strongly than the precipitated NM-200 and triggered a more evident inflammatory response, evaluated with Nos2 induction, NO production and the secretion of TNF-α, IL-6 and IL-1β. Moreover, both ASNP synergized macrophage activation by bacterial lipopolysaccharide (LPS), with a higher effect observed for NM-203. In conclusion, the results presented here demonstrate that, compared to precipitated, pyrogenic ASNP exhibit enhanced interaction with serum proteins and cell membrane, and cause a larger oxidative stress and stronger proinflammatory effects in macrophages. Therefore, these 2 nanomaterials should not be considered biologically equivalent.

Glutamine stimulates mTORC1 independent of the cell content of essential amino acids

Glutamine and leucine are important mTORC1 modulators, although their roles are not precisely defined. In HepG2 and HeLa cells glutamine-free incubation lowers mTORC1 activity, although cell leucine is not decreased. mTORC1 activity, suppressed by amino acid-free incubation, is completely rescued only if essential amino acids (EAA) and glutamine are simultaneously restored, although cell leucine is higher in the absence than in the presence of glutamine. Thus, glutamine stimulates mTORC1 independent of cell leucine, suggesting the existence of two distinct stimulatory signals from either glutamine or EAA.

The inhibition of glutamine synthetase sensitizes human sarcoma cells to l -asparaginase

PurposeTo evaluate the activity of the antitumor enzyme l-asparaginase (ASNase) on tumor cells of mesenchymal origin and the contribution of glutamine synthetase (GS) to the adaptation to the metabolic stress caused by the anti-tumor enzyme.MethodsWe studied the effects of ASNase in six human sarcoma cell lines: HT1080 (fibrosarcoma); RD (rhabdomyosarcoma); SW872 (liposarcoma); HOS, SAOS-2, and U2OS (osteosarcoma) in the absence or in the presence of the GS inhibitor methionine l-sulfoximine (MSO).ResultsHT1080 and SW872 cells were highly sensitive to ASNase-dependent cytotoxicity. In contrast, RD, SAOS-2, HOS, and U2OS cells exhibited only a partial growth suppression upon treatment with the anti-tumor enzyme. In these cell lines ASNase treatment was associated with increased levels of GS. When ASNase was used together with MSO, the proliferation of the poorly sensitive cell lines was completely blocked and a significant decrease in the IC50 for ASNase was observed. Moreover, when ASNase treatment was carried on in the presence of MSO, HOS and U2OS osteosarcoma cells exhibited a marked cytotoxicity, with increased apoptosis.ConclusionsIn human sarcoma cells (1) GS markedly contributes to the metabolic adaptation of tumor cells to ASNase and (2) the inhibition of GS activity enhances the antiproliferative and cytotoxic effects of ASNase. The two-step interference with glutamine metabolism, obtained through the combined treatment with ASNase and MSO, may provide a novel therapeutic approach that should be further investigated in human tumors of mesenchymal origin.

Glutamine depletion by crisantaspase hinders the growth of human hepatocellular carcinoma xenografts

Background:A subset of human hepatocellular carcinomas (HCC) exhibit mutations of β-catenin gene CTNNB1 and overexpress Glutamine synthetase (GS). The CTNNB1-mutated HCC cell line HepG2 is sensitive to glutamine starvation induced in vitro with the antileukemic drug Crisantaspase and the GS inhibitor methionine-L-sulfoximine (MSO).Methods:Immunodeficient mice with subcutaneous xenografts of the CTNNB1-mutated HCC cell lines HepG2 and HC-AFW1 were treated with Crisantaspase and/or MSO, and tumour growth was monitored. At the end of treatment, tumour weight and histology were assessed. Serum and tissue amino acids were determined by HPLC. Gene and protein expression were estimated with RT-PCR and western blot and GS activity with a colorimetric method. mTOR activity was evaluated from the phosphorylation of p70S6K1.Results:Crisantaspase and MSO depleted serum glutamine, lowered glutamine in liver and tumour tissue, and inhibited liver GS activity. HepG2 tumour growth was significantly reduced by either Crisantaspase or MSO, and completely suppressed by the combined treatment. The combined treatment was also effective against xenografts of the HC-AFW1 cell line, which is Crisantaspase resistant in vitro.Conclusions:The combination of Crisantaspase and MSO reduces glutamine supply to CTNNB1-mutated HCC xenografts and hinders their growth.

PKC-dependent stimulation of EAAT3 glutamate transporter does not require the integrity of actin cytoskeleton

The activity and the membrane expression of EAAT3 glutamate transporter are stimulated upon PKC activation by phorbol esters in C6 rat glioma cells. To investigate the role of cytoskeleton in these effects, we have employed actin-perturbing toxins and found that the perturbation of actin cytoskeleton inhibits basal but not phorbol-stimulated EAAT3 activity and membrane trafficking. In the absence of phorbols, latrunculin A, a toxin that disassembles actin cytoskeleton, produced a rapid inhibition of EAAT3 activity, due to a decrease in transport V(max). The inhibitory effect was fully reversible and was not detected for other sodium dependent transport systems for amino acids. However, latrunculin did not prevent the increase in transport caused by phorbol esters and, moreover, cells pre-treated with phorbols were resistant to the inhibitory effect of the toxin on EAAT3 activity. Biotinylation experiments indicated that the inhibitory effect of latrunculin was attributable to a decreased expression of the carrier on the membrane, while the toxin did not suppress the PKC-dependent increase in EAAT3 membrane abundance. Latrunculin A effects on EAAT3 were shared by cytochalasin D, a toxin that disorganizes actin filaments with a distinct mechanism of action. On the contrary, a small, but significant, increase of EAAT3 activity was observed upon incubation with jasplakinolide, a drug that stabilizes actin microfilaments. Also jasplakinolide, however, did not hinder phorbol-dependent stimulation of aspartate transport. Colchicine, a toxin that disrupts microtubules, also lowered EAAT3 activity without preventing transport stimulation by phorbols, while microtubule stabilization by paclitaxel led to an increase in aspartate transport. It is concluded that, in C6 cells, the PKC-mediated stimulatory effects on EAAT3 are cytoskeleton-independent, while in the absence of phorbols, the transporter is partially inhibited by the disorganization of either actin microfilaments or microtubules. These results suggest that EAAT3 trafficking in C6 cells involves different pools of transporters.

Imogolite: an aluminosilicate nanotube endowed with low cytotoxicity and genotoxicity.

High-aspect-ratio nanomaterials (HARN) (typically, single-walled carbon nanotubes (SWCNT) or multiwalled carbon nanotubes (MWCNT)) impair airway barrier function and are toxic to macrophages. Here, we assess the biological effects of nanotubes of imogolite (INT), a hydrated alumino-silicate [(OH)3Al2O3SiOH] occurring as single-walled NT, on murine macrophages and human airway epithelial cells. Cell viability was assessed with resazurin. RT-PCR was used to study the expression of Nos2 and Arg1, markers of classical or alternative macrophage activation, respectively, and nitrite concentration in the medium was determined to assess NO production. Epithelial barrier integrity was evaluated from the trans-epithelial electrical resistance (TEER). Potential genotoxicity of INT was assessed with comet and cytokinesis-block micronucleus cytome assays. Compared to MWCNT and SWCNT, INT caused much smaller effects on RAW264.7 and MH-S macrophage viability. The incubation of macrophages with INT at doses as high as 120 μg/cm(2) for 72 h did not alter either Nos2 or Arg1 expression nor did it increase NO production, whereas IL6 was induced in RAW264.7 cells but not in MH-S cells. INT did not show any genotoxic effect in RAW264.7 and A549 cells except for a decrease in DNA integrity observed in epithelial A549 cells after treatment with the highest dose (80 μg/cm(2)). No significant change in permeability was recorded in Calu-3 epithelial cell monolayers exposed to INT, whereas comparable doses of both SWCNT and MWCNT lowered TEER. Thus, in spite of their fibrous nature, INT appear not to be markedly toxic for in vitro models of lung-blood barrier cells.

Airway barrier dysfunction induced by exposure to carbon nanotubes in vitro: which role for fiber length?

Although carbon nanotubes (CNTs) are increasingly used, their biological effects are only incompletely characterized. However, experimental evidence suggests that the intratracheal instillation of CNTs causes the formation of interstitial granulomas and progressive pulmonary fibrosis in rodents. Using human epithelial Calu-3 cells as a model of airway epithelium in vitro, we have recently reported that the exposure to commercial multi-walled CNTs (MWCNTs) causes a progressive decrease of the transepithelial electrical resistance (TEER), pointing to a CNT-dependent impairment of the epithelial barrier function. To characterize better this behavior, we compared the effects of two types of MWCNTs and single-walled CNTs (SWCNTs) of different lengths on the TEER of Calu-3 monolayers. All the materials were used at a dose of 100 μg/mL corresponding to an exposure of 73 μg/cm2 of cell monolayer. Only the longer MWCNTs and SWCNTs cause a significant decrease in TEER. To elucidate the mechanism underlying the changes in barrier function, the expression of the junction proteins occludin and ZO-1 has been also assessed. No significant decrease in the mRNA for either protein is detectable after the exposure to any type of CNTs. It is concluded that the impairment of barrier function in Calu-3 monolayers is a peculiar effect of CNTs endowed with clear cut fiber properties and is not referable to marked changes in the expression of junction proteins.