Maria Francesca Aleo

Stress proteins and oxidative damage in a renal derived cell line exposed to inorganic mercury and lead

A close link between stress protein up-regulation and oxidative damage may provide a novel therapeutic tool to counteract nephrotoxicity induced by toxic metals in the human population, mainly in children, of industrialized countries. Here we analysed the time course of the expression of several heat shock proteins, glucose-regulated proteins and metallothioneins in a rat proximal tubular cell line (NRK-52E) exposed to subcytotoxic doses of inorganic mercury and lead. Concomitantly, we used morphological and biochemical methods to evaluate metal-induced cytotoxicity and oxidative damage. In particular, as biochemical indicators of oxidative stress we detected reactive oxygen species (ROS) and nitrogen species (RNS), total glutathione (GSH) and glutathione-S-transferase (GST) activity. Our results clearly demonstrated that mercury increases ROS and RNS levels and the expressions of Hsp25 and inducible Hsp72. These findings are corroborated by evident mitochondrial damage, apoptosis or necrosis. By contrast, lead is unable to up-regulate Hsp72 but enhances Grp78 and activates nuclear Hsp25 translocation. Furthermore, lead causes endoplasmic reticulum (ER) stress, vacuolation and nucleolar segregation. Lastly, both metals stimulate the over-expression of MTs, but with a different time course. In conclusion, in NRK-52E cell line the stress response is an early and metal-induced event that correlates well with the direct oxidative damage induced by mercury. Indeed, different chaperones are involved in the specific nephrotoxic mechanism of these environmental pollutants and work together for cell survival.

Cisplatin triggers atrophy of skeletal C2C12 myotubes via impairment of Akt signalling pathway and subsequent increment activity of proteasome and autophagy systems.

Cisplatin (cisPt) is an antineoplastic drug which causes an array of adverse effects on different organs and tissues, including skeletal muscle. In this work we show that cisPt behaves as a potent trigger to activate protein hypercatabolism in skeletal C2C12 myotubes. Within 24h of 50 μM cisPt administration, C2C12 myotubes displayed unchanged cell viability but showed a subset of hallmark signs typically recognized during atrophy, including severe reduction in body size, repression of Akt phosphorylation, transcriptional up-regulation of atrophy-related genes, such as atrogin-1, gabarap, beclin-1 and bnip-3, and loss of myogenic markers. As a consequence, proteasomal activity and formation of autophagosomes were remarkably increased in cisPt-treated myotubes, but forced stimulation of Akt pathway, as obtained through insulin administration or delivery of a constitutively activated Akt form, was sufficient to counter the cisPt-induced protein breakdown, leading to rescue of atrophic size. Overall, these results indicate that cisPt induces atrophy of C2C12 myotubes via activation of proteasome and autophagy systems, suggesting that the Akt pathway represents one sensitive target of cisPt molecular action in skeletal muscle.

Antioxidant potential and gap junction-mediated intercellular communication as early biological markers of mercuric chloride toxicity in the MDCK cell line.

In this study, the early nephrotoxic potential of mercuric chloride (HgCl(2)) has been evaluated in vitro, by exposing a renal-derived cell system, the tubular epithelial Madin-Darby canine kidney (MDCK) cell line, to the presence of increasing HgCl(2) concentrations (0.1-100 microM) for different periods of time (from 4 to 72 h). As possible biological markers of the tubular-specific toxicity of HgCl(2) in exposed-MDCK cultures we analysed: (i) critical biochemical parameters related to oxidative stress conditions and (ii) gap-junctional function (GJIC). HgCl(2) cytotoxicity was evaluated by cell-density assay. The biochemical analysis of the pro-oxidant properties of the mercuric ion (Hg(2+)) was performed by evaluating the effect of the metal salt on the antioxidant status of the MDCK cells. The cell glutathione (GSH) content and the activity of glutathione peroxidase (Gpx) and catalase (Cat), two enzymes engaged in the H(2)O(2) degradation, were quantified. HgCl(2) influence on MDCK GJIC was analysed by the microinjection/dye-transfer assay. HgCl(2)-induced morphological changes in MDCK cells were also taken into account. Our results, proving that subcytotoxic (0.1-10 microM) HgCl(2) concentrations affect either the antioxidant defences of MDCK cells or their GJIC, indicate these critical functions as suitable biological targets of early mercury-induced tubular cell injury.

Synthesis, spectral properties and enzymatic hydrolysis of fluorescent derivatives of cerebroside sulfate containing long-wavelength-emission probes

Fluorescent derivatives of cerebroside sulfate (sulfogalactosyl ceramide, sulfatide) containing long-wavelength-emission fluorophores were synthesized. For this purpose a procedure was developed for preparing a cerebroside 3-sulfate derivative with an amino group on the terminal carbon atom of its fatty acyl residue. The latter compound has been used to prepare cerebroside 3-sulfate, coupled to lissamine-rhodamine, fluoresceine, eosine and NBD. The spectroscopic properties of these compounds, in different solvent systems and when incorporated into micelles of a non-ionic detergent or liposomes of a phospholipid, are reported. Incubation of these respective sulfatides with a human leukocyte preparation, resulted in the formation of the corresponding fluorescent cerebrosides.

Uptake and metabolism of a fluorescent sulfatide analogue in cultured skin fibroblasts

The sulfatide fluorescent analogue N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulfate was administered in the form of albumin complex to normal human skin fibroblasts and its metabolic fate was investigated. Ceramide, galactosylceramide, glucosylceramide, sphingomyelin and free acid, all containing the fluorophore lissamine rhodamine, have been synthesized as reference standards for the identification of the metabolic products. Ceramide appeared to be the main metabolic product present both in cell extract and medium, followed by galactosylceramide and sphingomyelin. Fluorescence microscopy of cells showed a marked perinuclear fluorescence.

Focal adhesion molecules as potential target of lead toxicity in NRK-52E cell line.

In this study, we investigated the influence of inorganic lead (Pb(II)), an environmental pollutant having nephrotoxic action, on the focal adhesion (FA) organization of a rat kidney epithelial cell line (NRK‐52E). In particular, we evaluated the effects of the metal on the recruitment of paxillin, focal adhesion kinase, vinculin and cytoskeleton proteins at the FAs complexes. We provided evidences that, in proliferating NRK‐52E cell cultures, low concentrations of Pb(II) affect the cell adhesive ability and stimulate the disassembly of FAs, thus inhibiting the integrin‐activated signalling. These effects appeared to be strictly associated to the Pb‐induced arrest of cell cycle at G0/G1 phase also proved in this cell line.

Different role of Schisandrin B on mercury-induced renal damage in vivo and in vitro

Mercuric chloride (HgCl₂) causes acute oxidant renal failure that affects mainly proximal tubules. Schisandrin B (Sch B), an active lignan from the fruit of Schisandra chinensis, has been successfully used to treat gentamicin nephrotoxicity, but its role against mercury damage is still largely unknown. Here we analysed in vivo and in vitro the efficacy of Sch B supplementation against HgCl₂ nephrotoxicity, focusing on histopathology, stress proteins, oxidative (cytochrome c oxidase) and nitrosactive markers (eNOS, nNOS). Wistar rats were treated with Sch B (10 mg/kg/day p.o.) or vehicle (olive oil) for 9 days, then coadministered with a single HgCl₂ nephrotoxic dose (3.5 mg/kg i.p.) and killed after 24 h. The tubular and mitochondrial damage induced by mercury was limited by Sch B coadministration in vivo. Remarkably, after Sch B and mercury challenge, HSP25, HSP72, GRP75 were reduced in the renal cortex, cytochrome c oxidase increased and eNOS and nNOS were restored in glomeruli. In contrast, NRK-52E proximal tubular cells treated with Sch B 6.25 μM plus HgCl₂ 20 μM did not show any amelioration on viability and oxidative stress in respect to HgCl₂ 20 μM alone. Moreover, after Sch B plus mercury in vitro treatment, HSP72 staining persisted while HSP25 further increased. Thus, in our experimental conditions, Sch B cotreatment afforded better protection against mercury poisoning in vivo than in vitro. This discrepancy might be partly attributable to Sch B influence on glomerular perfusion as corroborated by the recovery of vasoactive markers like macular and endothelial nitric oxide isoforms.

Rapid preparation of a distinct lysosomal population from myelinating mouse brain using percoll gradients

Abstract: To study the vesicular lysosome‐associated transport and the metabolism of some brain macromolecules (in particular, sialoglycoconjugates), we developed a rapid procedure to obtain a distinct lysosomal population starting from myelinating mouse brain. This procedure is based on an initial differential centrifugation step producing a 1,000–17,500‐g fraction (P2), followed by isopycnic centrifugation of fraction P2 on a self‐generated colloidal silica gel (Percoll) gradient. The heaviest subfraction thus obtained is very rich in acid hydrolase activities like β‐galactosidase, arylsulfatase A, and acid phosphatase. The enrichment of these enzymes is ∼100‐fold as compared with the starting homogenate, whereas the markers of other subcellular organelles, such as mitochondria, plasma membranes, or the Golgi apparatus, are virtually absent. The lysosomal preparation contains ∼12–14% of the total acid hydrolase activities, with a protein yield of ∼0.12%. Electron microscopy shows that the lysosomal fraction is composed of an ∼90% pure population of lysosomes. Therefore, the procedure described here is suitable for obtaining a highly purified lysosome preparation from myelinating mouse brain.

Cobalt triggers necrotic cell death and atrophy in skeletal C2C12 myotubes

Severe poisoning has recently been diagnosed in humans having hip implants composed of cobalt-chrome alloys due to the release of particulate wear debris on polyethylene and ceramic implants which stimulates macrophagic infiltration and destroys bone and soft tissue, leading to neurological, sensorial and muscular impairments. Consistent with this premise, in this study, we focused on the mechanisms underlying the toxicity of Co(II) ions on skeletal muscle using mouse skeletal C2C12 myotubes as an in vitro model. As detected using propidium iodide incorporation, increasing CoCl2 doses (from 5 to 200μM) affected the viability of C2C12 myotubes, mainly by cell necrosis, which was attenuated by necrostatin-1, an inhibitor of the necroptotic branch of the death domain receptor signaling pathway. On the other hand, apoptosis was hardly detectable as supported by the lack of caspase-3 and -8 activation, the latter resulting in only faint activation after exposure to higher CoCl2 doses for prolonged time points. Furthermore, CoCl2 treatment resulted in atrophy of the C2C12 myotubes which was characterized by the increased expression of HSP25 and GRP94 stress proteins and other typical `pro-atrophic molecular hallmarks, such as early activation of the NF-kB pathway and down-regulation of AKT phosphorylation, followed by the activation of the proteasome and autophagy systems. Overall, these results suggested that cobalt may impact skeletal muscle homeostasis as an inducer of cell necrosis and myofiber atrophy.

Metabolic fate of extracellular NAD in human skin fibroblasts

Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface‐located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD‐glycohydrolase, nucleotide pyrophosphatase and 5′‐nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5′‐nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U‐[14C]‐adenine‐NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto‐NAD‐hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with 14C‐NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [14C]‐NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with 14C‐adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [14C]‐adenosine yield the same radioactive products as with [14C]‐NAD; the absence of inhibition of [14C]‐adenosine uptake by ADPribose in the presence of α‐β methyleneADP, an inhibitor of 5′ nucleotidase, demonstrates that ADPribose coming from NAD via NAD‐glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent. J. Cell. Biochem. 80:360–366, 2001.