MC3T3-E1 pre-osteoblast response and differentiation after graphene oxide nanosheet uptake.

Nano-graphene oxide (GO) and its functionalized derivatives have aroused a great interest for drug delivery, tissue engineering and photothermal cancer therapy, but their biocompatibility has not yet been fully assessed. The aim of the present study was to evaluate the proliferation and differentiation of MC3T3-E1 pre-osteoblasts after the uptake of GO nanosheets (c.a. 400nm), functionalized with poly(ethylene glycol-amine) (PEG) and labelled with fluorescein isothiocyanate (FITC). Significant proliferation decrease and apoptosis increase were observed 3days after incorporation of FITC-PEG-GO by MC3T3-E1 cells. However, alterations on healthy pre-osteoblast differentiation into cells exhibiting osteoblast phenotype were not observed, as they showed normal alkaline phosphatase levels and matrix mineralization 12days after nanosheet uptake. The results suggest that 40µg/mL concentrations of these nanosheets would not affect the differentiation of healthy pre-osteoblasts, thus these PEG-GO nanosheets have potential to be used for biomedical applications after their internalization, as the induction of local hyperthermia on bone cancer.

Integrated biomimetic carbon nanotube composites for in vivo systems.

As interest in using carbon nanotubes for developing biologically compatible systems continues to grow, biological inspiration is stimulating new directions for in vivo approaches. The ability to integrate nanotechnology-based systems in the body will provide greater successes if the implanted material is made to mimic elements of the biological milieu especially through tuning physical and chemical characteristics. Here, we demonstrate the highly successful capacity for in vivo implantation of a new carbon nanotube-based composite that is, itself, integrated with a hydroxyapatite-polymethyl methacrylate to create a nanocomposite. The success of this approach is grounded in finely tailoring the physical and chemical properties of this composite for the critical demands of biological integration. This is accomplished through controlling the surface modification scheme, which affects the interactions between carbon nanotubes and the hydroxyapatite-polymethyl methacrylate. Furthermore, we carefully examine cellular response with respect to adhesion and proliferation to examine in vitro compatibility capacity. Our results indicate that this new composite accelerates cell maturation through providing a mechanically competent bone matrix; this likely facilitates osteointegration in vivo. We believe that these results will have applications in a diversity of areas including carbon nanotube, regeneration, chemistry, and engineering research.

Life and death in aluminium-exposed cultures of rat lactotrophs studied by flow cytometry.

Prolonged exposure to aluminium may impact health. Aluminium's deleterious effects are mostly attributed to its selective accumulation in particular organs and cell types. Occupational exposure to aluminium is allied with a reduced level of serum prolactin, a stress peptide hormone mainly synthesised and secreted by the anterior pituitary lactotrophs. Our aim was to study the effect of aluminium on the viability of rat lactotrophs in primary suspension cultures where multicellular aggregates tend to form, comprising approximately two thirds of the total cell population as confirmed by confocal microscopy. Flow cytometric light scattering of calcein acetoxymethyl ester and ethidium homodimer-1 labelled cells was used to define subpopulations of live and dead cells in heterogeneous suspensions comprised of single cells and multicellular aggregates of distinct size. Concentration-dependent effects of AlCl(3) were observed on aggregate size and cell survival. After 24-h exposure to 3 mM AlCl(3), viability of single cells declined from 5% to 3%, while in multicellular aggregates, viability declined from 23% to 20%. The proportion of single cells increased from 30% to 42% within the same concentration range, while in large aggregates, the proportion remained approximately constant representing 35% of the cell suspension. In large aggregates, cell viability (75%) remained unaltered after exposure to AlCl(3) concentrations up to 300 microM, while in single cells, viability was halved at 30 microM. In conclusion, our finding indicates that prolonged exposure to aluminium may lead to significant loss of pituitary cells.

Comparative effects of aluminum and ouabain on synaptosomal choline uptake, acetylcholine release and (Na+/K+)ATPase.

Closing the gap between adverse health effects of aluminum and its mechanisms of action still represents a huge challenge. Cholinergic dysfunction has been implicated in neuronal injury induced by aluminum. Previously reported data also indicate that in vivo and in vitro exposure to aluminum inhibits the mammalian (Na(+)/K(+))ATPase, an ubiquitous plasma membrane pump. This study was undertaken with the specific aim of determining whether in vitro exposure to AlCl(3) and ouabain, the foremost utilized selective inhibitor of (Na(+)/K(+))ATPase, induce similar functional modifications of cholinergic presynaptic nerve terminals, by comparing their effects on choline uptake, acetylcholine release and (Na(+)/K(+))ATPase activity, on subcellular fractions enriched in synaptic nerve endings isolated from rat brain, cuttlefish optic lobe and torpedo electric organ. Results obtained show that choline uptake by rat synaptosomes was inhibited by submillimolar AlCl(3), whereas the amount of choline taken up by synaptosomes isolated from cuttlefish and torpedo remained unchanged. Conversely, choline uptake was reduced by ouabain to a large extent in all synaptosomal preparations analyzed. In contrast to ouabain, which modified the K(+) depolarization evoked release of acetylcholine by rat, cuttlefish and torpedo synaptosomal fractions, AlCl(3) induced reduction of stimulated acetylcholine release was only observed when rat synaptosomes were challenged. Finally, it was observed that the aluminum effect on cuttlefish and torpedo synaptosomal (Na(+)/K(+))ATPase activity was slight when compared to its inhibitory action on mammalian (Na(+)/K(+))ATPase. In conclusion, inhibition of (Na(+)/K(+))ATPase by AlCl(3) and ouabain jeopardized the high-affinity (Na(+)-dependent, hemicholinium-3 sensitive) uptake of choline and the Ca(2+)-dependent, K(+) depolarization evoked release of acetylcholine by rat, cuttlefish and torpedo synaptosomal fractions. The effects of submillimolar AlCl(3) on choline uptake and acetylcholine release only resembled those of ouabain when rat synaptosomes were assayed. Therefore, important differences were found between the species regarding the cholinotoxic action of aluminum. The variability of (Na(+)/K(+))ATPase sensitivity to aluminum of cholinergic neurons might contribute to their differential susceptibility to this neurotoxic agent.

Effect of chronic exposure to aluminium on isoform expression and activity of rat (Na+/K+)ATPase.

The ability of aluminum to inhibit the (Na(+)/K(+))ATPase activity has been observed by several investigators. The (Na(+)/K(+))ATPase is characterized by a complex molecular heterogeneity that results from the expression and differential association of multiple isoforms of both catalytic (alpha) and regulatory (beta) subunits. For instance, three main alpha (alpha(1), alpha(2) and alpha(3)) and three beta (beta(1), beta(2) and beta(3)) subunit isoforms exist in vertebrate nervous tissue, whereas only alpha(1) and beta(1) have been identified in kidney. However, no studies have focused on determining the change in (Na(+)/K(+))ATPase isoforms caused by chronic exposure to aluminum and its relation with aluminum toxicity. In this study, adult male Wistar rats were submitted to chronic dietary AlCl(3) exposure (0.03 g/day of AlCl(3) for 4 months), and the activity and protein expression of (Na(+)/K(+))ATPase isozymes were studied in brain cortex synaptosomes and in kidney homogenates. The intracellular levels of adenine nucleotides, plasma membrane integrity, and aluminum accumulation were also studied in brain synaptosomes. Aluminum accumulation upon chronic dietary AlCl(3) administration significantly decreased the (Na(+)/K(+))ATPase activity measured in the presence of nonlimiting Mg-ATP concentrations, without compromising protein expression of alpha-subunit isoforms in brain and kidney. Aluminum-induced synaptosomal (Na(+)/K(+))ATPase inhibition was due to a reduction in the activity of isozymes containing alpha(1)-alpha(2) and alpha(3)-subunits. The onset of enzyme inhibition was accompanied by a decrease of the (Na(+)/K(+))ATPase sensitivity to submicromolar concentrations of ouabain, and it preceded major damage in plasma membrane integrity and energy supply, as revealed by the analysis of lactate dehydrogenase leakage and endogenous adenine nucleotides. The data suggest that, during chronic dietary exposure to AlCl(3), brain (Na(+)/K(+))ATPase activity drops, even if no significant alterations of catalytic subunit protein expression, cellular energy depletion, and changes in cell membrane integrity are observed. Implications regarding underlying mechanisms of aluminum neurotoxicity are discussed.

The inhibitory effect of aluminium on the (Na+/K+)ATPase activity of rat brain cortex synaptosomes.

The effect of AlCl(3) on the (Na(+)/K(+))ATPase activity of freeze-thawed synaptosomes, isolated from rat brain cortex, has been studied. The AlCl(3) action on the enzyme hydrolytic activity was examined using in vitro and in vivo approaches. Following exposure to AlCl(3) using both in vitro (synaptosomes incubated in the presence of AlCl(3) for 5 min) and in vivo (synaptosomes isolated from rats that received 0.03 g AlCl(3)/day for 4 months) approaches, the (Na(+)/K(+))ATPase activity was inhibited in a concentration-dependent way. The maximal inhibitory effect (approximately 60%) was observed in the presence of a AlCl(3) concentration >75 microM and at non-limiting ATP concentrations. Conversely, AlCl(3) did not inhibit the enzyme activity when UTP was used as substrate instead of ATP. Analysis of the substrate dependence of membrane-bound (Na(+)/K(+))ATPase by a computer simulation model suggests that the AlCl(3)-induced inhibitory effect is characterised by a reduction of the rate-limiting step velocity of the reaction cycle. Moreover, it seems that aluminium can induce impairment of the interprotomeric interaction within the oligomeric ensemble of membrane-bound (Na(+)/K(+))ATPase. In fact, this effect was accompanied by a slight, but significant, decrease of readily accessible SH groups, which are involved in the maintenance of the membrane-bound (Na(+)/K(+))ATPase oligomeric structure. In conclusion, during exposure to aluminium, reduction of the activation of membrane-bound (Na(+)/K(+))ATPase by high ATP concentrations occurs, which results in a partial inhibition of the enzyme.

Aluminum accumulation and membrane fluidity alteration in synaptosomes isolated from rat brain cortex following aluminum ingestion: effect of cholesterol.

In the present work, we studied the effect of cholesterol/phospholipid (CH/PL) molar ratio on aluminum accumulation and aluminum-induced alteration of membrane fluidity in rat brain cortex synaptosomes. We observed that sub-acute (daily supply of 1.00 g of AlCl(3) during 10 days) and chronic (daily supply of 0.03 g of AlCl(3) during 4 months) exposure to dietary aluminum leads to a synaptosomal aluminum enrichment of 45 and 59%, respectively. During chronic exposure to AlCl(3), the enhancement of aluminum content was prevented by administration of colestipol (0.31 g/day), which decreased the synaptosomal membrane CH/PL molar ratio (nmol/nmol) from 1.2 to 0.4. Fluorescence anisotropy analysis, using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene (TMA-DPH), showed that after treatment with colestipol a decrease in membrane order occurs at the level of hydrophilic lipid-water surface and deeper hydrophobic region of the synaptosomal membrane. When the rats were exposed to aluminum, it was observed a significant enhancement of membrane fluidity, which was more pronounced at the level of the membrane hydrophilic regions. Meanwhile, when chronic exposure to dietary AlCl(3) was accompanied by treatment with colestipol, the aluminum-induced decrease in membrane order was negligible when compared to TMA-DPH and DPH anisotropy values measured upon colestipol treatment. In contrast, in vitro incubation of synaptosomes (isolated from control rats) with AlCl(3) induced a concentration-dependent rigidification of this more hydrophilic membrane region. The opposite action of aluminum on synaptosomal membrane fluidity, during in vivo and in vitro experiments, appears to be explained by alteration of synaptosomal CH/PL molar ratio, since a significant reduction (approximately 80%) of this parameter occurs during in vivo exposure to aluminum. In conclusion, during in vivo exposure to aluminum, fluidification of hydrophilic regions and reduction of CH/PL molar ratio of presynaptic membranes accompany the accumulation of this cation, which appear to restrict aluminum retention in brain cortex nerve terminals.