Abstract: A major source of the wide presence of EDCs (Endocrine Disrupting Compounds) in water bodies is represented by direct/indirect discharge of sewage. Recent scientific literature reports data about their trace concentration in water, sediments and aquatic organisms, as well as removal efficiencies of different wastewater treatment schemes. Despite the availability of a huge amount of data, some doubts still persist due to the difficulty in evaluating synergistic effects of trace pollutants in complex matrices. In this paper, an integrated assessment procedure was used, based on chemical and biological analyses, in order to compare the performance of two full scale biological wastewater treatment plants (either equipped with conventional settling tanks or with an ultrafiltration membrane unit) and tertiary ozonation (pilot scale). Nonylphenol and bisphenol A were chosen as model EDCs, together with the parent compounds mono- and di-ethoxylated nonylphenol (quantified by means of GC-MS). Water estrogenic activity was evaluated by applying the human breast cancer MCF-7 based reporter gene assay. Process parameters (e.g., sludge age, temperature) and conventional pollutants (e.g., COD, suspended solids) were also measured during monitoring campaigns. Conventional activated sludge achieved satisfactory removal of both analytes and estrogenicity. A further reduction of biological activity was exerted by MBR (Membrane Biological Reactor) as well as ozonation; the latter contributed also to decrease EDC concentrations.
Abstract: The development and validation of reliable in vitro methods alternative to conventional in vivo studies in experimental animals is a well-recognised priority in the fields of pharmaco-toxicology and food research. Conventional studies based on two-dimensional (2-D) cell monolayers have demonstrated their significant limitations: the chemically and spatially defined three-dimensional (3-D) network of extracellular matrix components, cell-to-cell and cell-to-matrix interactions that governs differentiation, proliferation and function of cells in vivo is, in fact, lost under the simplified 2-D condition. Being able to reproduce specific tissue-like structures and to mimic functions and responses of real tissues in a way that is more physiologically relevant than what can be achieved through traditional 2-D cell monolayers, 3-D cell culture represents a potential bridge to cover the gap between animal models and human studies. This article addresses the significance and the potential of 3-D in vitro systems to improve the predictive value of cell-based assays for safety and risk assessment studies and for new drugs development and testing. The crucial role of tissue engineering and of the new microscale technologies for improving and optimising these models, as well as the necessity of developing new protocols and analytical methods for their full exploitation, will be also discussed.
Abstract: Skeletal abnormalities reported in humans and laboratory animals after spaceflight, include cancellous osteopenia, decreased cortical and cancellous bone formation, aberrant matrix ultrastructure, decreased mineralization and reduced bone strength. Although considerable effort has been made up to now to understand the skeletal effects of spaceflight, in order to estimate health risk, our knowledge in this area is still largely incomplete. It is widely accepted that the mechanical strength of cancellous bone is related not only to the mineral content, but also to the trabecular micro-architecture arrangement. Three-dimensional numerical analysis of bone volumes has been shown to be an important tool in this field. The Cell Method, a recently introduced numerical method, has been applied to static analysis of structures obtained from 3D reconstruction of micro-computed tomography scans performed at the Elettra Synchrotron facility (Trieste, Italy) in order to quantify changes in trabecular bone architecture. In the present study, the Cell Method model is used to compare the micro-tomographed structure of fragments of rats bone explants (tibial proximal epiphyses) harvested after 3 days and after 1, 2, 3 and 4 weeks of culture in the RCCS bioreactor, which represents the unique existing bioreactor, operating on the Earth's surface, capable of successfully reproducing, in vitro, optimal conditions in order to simulate a microgravity environment. Although preliminary, our results seem to suggest that the exposure of tibial bone explants to simulated microgravity conditions obtained by the RCCS bioreactor, are consistent with skeletal changes observed after spaceflight.
Abstract: Major hepatic resection in cirrhotic patients is associated with impaired liver regeneration and failure, leading to high peri-operative mortality. In this work, the causes of defective regeneration in cirrhotic liver and the utility of IL-6 treatment were investigated in an experimental model combining cirrhosis and partial hepatectomy in the rat. Relative to normal controls, decompensated cirrhotic animals showed decreased survival, while compensated cirrhotic animals showed similar survival but reduced hepatic DNA synthesis and newly regenerated liver mass amount. Defective liver regeneration was associated with a decrease in STAT3 and NF-kB activation, consistent with an increased accumulation of their respective inhibitors PIAS3 and IkBalpha, and with a decreased induction of Bcl-xL. Treatment with recombinant IL-6 enhanced survival of decompensated cirrhotic animals, while it did not affect survival of compensated cirrhotic animals but sustained liver regeneration, by restoring STAT3 and NF-kB activation and Bcl-xL induction to the levels found in normal controls. The pro-growth effects exerted by IL-6 treatment in cirrhotic liver were attained also at low, pharmacologically acceptable doses. In conclusion, our results suggest that IL-6 treatment may be therapeutic in major resection of cirrhotic liver.
Abstract: Manganese (Mn) is both an essential nutrient and a toxicant, with specific effects on liver and kidney (acute exposure) and on central nervous system (CNS) (chronic exposure). Mn neurotoxicity includes neurobehavioral disorders and extra-pyramidal motor dysfunctions (manganism), possibly due to focal injuries to the basal ganglia. Even if widely investigated, the molecular mechanisms responsible for Mn toxicity remain to be clarified. Aim of this study was to identify suitable in vitro models to investigate these molecular pathways. To this purpose we compared the effect of manganese chloride on four cell lines, representative of the main target organs of Mn toxicity in vivo. HepG2 and MDCK cell lines were selected for liver and kidney, respectively; glial GL15 and neuronal SHSY5Y cells were used as models of CNS components. To complete the "motor system" model, skeletal muscle C2C12 cells were also included. Our results demonstrate that hepatic, renal, glial and neuronal cell types differently react to Mn, mirroring the specific in vivo response of the tissue they represent. This confirms their value as suitable in vitro models to study Mn-related toxic events. Interestingly, also muscle C2C12 cells showed a noticeable sensitivity to Mn, preferential targets being differentiated myotubes.
Abstract: Relative insolubility of inorganic Pb compounds is one of the major problems in the evaluation of the toxicological profile of this metal. Different characteristics of Pb-containing solutions may, in fact, alter the biological properties of Pb compounds and influence their toxic potency. To investigate these aspects, we used selected experimental conditions to evaluate and compare the specific biological effects of five inorganic Pb compounds (soluble salts and oxide) on the viability and proliferation rate of a rat liver-derived cell line (REL cells). The study was performed according to classical toxicological criteria (dose- and time-response, reversibility/transience of the effect). Each Pb compound was accurately solubilised and the quantification of the real concentration of Pb(II) ions was performed either on the culture media used for each treatment, or on the extracts of exposed cells. Our study shows that four, out of the five Pb compounds we tested, induce the same dose- and time-related anti-proliferative effects on REL cells, being these effects also reversible, transient and directly related to the intracellular content of the metal. Since the intracellular concentration of the metal and, consequently, its biological effects on REL cells, directly depends on the bioavailability of the Pb(II) cation present in the treatment solutions, our results indicate that, in the experimental procedures aimed to assess the toxic potency of this metal, the solubility of each Pb compound should be carefully evaluated and taken into account.
Abstract: Tubular epithelium represents the primary target of mercuric ions (Hg(2+)) nephrotoxicity. Although widely investigated, the mechanisms of Hg(2+) cell uptake, accumulation and excretion all along the nephron remain largely unknown. In the present study, native distal tubular-derived Madin-Darby canine kidney (MDCK) cells exposed to subcytotoxic (micromolar) HgCl(2) concentrations were used for investigating specific mechanisms involved in the tubular response to toxic metals. Inductively coupled plasma-mass spectrometry (ICP-MS) was firstly used for assessing HgCl(2) solubility and then for quantifying Hg(2+) cell uptake. Exposed to HgCl(2), MDCK cells showed a rapid, but transient, Hg(2+) accumulation. The metallic cation was found to affect cell density and morphology, being these effects related to the dose and the time of exposure. In parallel, an Hg(2+)-induced up-regulation of endogenous MRP1 and MRP2 export pumps, a significant HgCl(2)-dependent induction of protective cellular thiols and an increase in the glutathione conjugates metabolism were also observed. The functional suppression of MRPs activity, obtained by MK-571 treatment, increased the Hg(2+) cell content and the sensitivity of MDCK cells to HgCl(2). Our results demonstrate that, in MDCK cells, inorganic Hg(2+) promotes the activation of specific detoxifying pathways that may, at least partly, depend on the activity of MRP transporters.
Abstract: Opposite effects of nuclear factor-kappaB (NF-kappaB) on neuron survival rely on activation of diverse NF-kappaB factors. While p65 is necessary for glutamate-induced cell death, c-Rel mediates prosurvival effects of interleukin-1beta. However, it is unknown whether activation of c-Rel-dependent pathways reduces neuron vulnerability to amyloid-beta (Abeta), a peptide implicated in Alzheimer's disease pathogenesis. We show that neuroprotection elicited by activation of metabotropic glutamate receptors type 5 (mGlu5) against Abeta toxicity depends on c-Rel activation. Abeta peptide induced NF-kappaB factors p50 and p65. The mGlu5 agonists activated c-Rel, besides p50 and p65, and the expression of manganese superoxide dismutase (MnSOD) and Bcl-X(L). Targeting c-Rel expression by RNA interference suppressed the induction of both antiapoptotic genes. Targeting c-Rel or Bcl-X(L) prevented the prosurvival effect of mGlu5 agonists. Conversely, c-Rel overexpression or TAT-Bcl-X(L) addition rescued neurons from Abeta toxicity. These data demonstrate that mGlu5 receptor activation promotes a c-Rel-dependent antiapoptotic pathway responsible for neuroprotection against Abeta peptide.
Abstract: We have previously shown that long-term thyroxine administration can protect the heart against ischemia. In the present study, we investigated whether thyroxine-induced cardioprotection can mimic the pattern of protection that is afforded by a well-established cardioprotective means such as ischemic preconditioning. In a Langendorff-perfused rat heart preparation, after an initial stabilization, normal and thyroxine-treated hearts were subjected to 20 minutes of zero-flow global ischemia followed by 45 minutes of reperfusion. In thyroxine-treated hearts, phospho-p38 mitogen-activated protein kinase (MAPK) was found to be less at the end of the ischemic period, whereas ischemic contracture was accelerated and postischemic recovery was increased in comparison to normal hearts. In addition, normal hearts were subjected to a four-cycle preconditioning protocol before ischemia. Phospho-p38 MAPK was found to be less at the end of the ischemic period in preconditioned hearts, whereas ischemic contracture was accelerated and postischemic functional recovery was increased in those hearts in comparison to nonpreconditioned hearts. An increase in basal expression and phosphorylation of PKCdelta was also found to occur after long-term thyroxine administration. We conclude that long-term thyroxine administration can protect the heart from ischemic injury through a pattern of protection that closely resembles that of ischemic preconditioning.
Abstract: 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.
Abstract: Mercury (Hg), one of the most diffused and hazardous organ-specific environmental contaminants, exists in a wide variety of physical and chemical states, each of which with unique characteristics of target organ specificity. Exposure to Hg vapour and to organic mercurials specifically affects the CNS, while the kidney is the target organ for inorganic Hg compounds. Despite the increasing number of studies, the molecular bases of the nephrotoxic potential of Hg has not, up to now, been clarified, even if there is evidence suggesting that the ability of the metal to interact with proteins (thiol groups) or to generate oxygen radicals may play a major role. Within this context, the aim of the present study was to investigate, in vitro, the mechanism(s) of the early nephrotoxic potential of mercury chloride (HgCl2), one of the most diffused and biologically active mercury (Hg2+) compounds. For this purpose, two kidney-derived in vitro systems (the MDCK and the LLC-PK1 cell lines) were tested for their sensitivity to the salt, and MDCK was chosen as the most suitable in vitro model for our study. As possible biological markers of the organ-specific toxicity of the metal we analysed: i) critical biochemical parameters related to oxidative stress conditions (effect of Hg2+ on the anti-oxidant status of the cell), and ii) gap-junctional function (GJIC).
Abstract: It has been proposed that GL15, a human cell line derived from glioblastoma multiforme, is a possible astroglial-like cell model, based on the presence of cytoplasmic glial fibrillary acidic protein.
Abstract: Immortalization of chondrocytes by SV40 T Ag has often been reported to trigger the loss of expression of type II collagen, one of the main differentiation markers, although some immortalized chondrocyte lines maintaining a differentiated phenotype have also been described. Here, we show using transient cotransfections in differentiated chondrocytes that, in contrast to c-src, neither SV40 T Ag, nor c-myc, decreases col2a1 transcriptional activity. Then, we report the possibility of immortalizing rabbit articular chondrocytes by expression of SV40 T Ag controlled by the col2a1 promoter and enhancer (pCol2SV). This strategy allows one to select within a population of differentiated chondrocytes those which are able to maintain functional regulation of the col2a1 gene through long-term culture. In precrisis pCol2SV-transfected chondrocytes, all-trans-retinoic acid, a down-regulator of col2a1 expression, induced apoptosis, strongly suggesting the strict control of T Ag expression by col2a1 regulatory sequences. Some pCol2SV-transfected chondrocytes were definitively immortalized, after a short crisis period. However, type II collagen synthesis was restricted to a small proportion of cells, which went on to decrease with subculture, while the proportion of cells expressing T Ag was not affected. In these postcrisis cells, T Ag remained at least partially under the control of functional col2a1 regulatory elements as assessed by all-trans-retinoic acid down-regulation.
Abstract: Chondrocytes cultivated in monolayer rapidly divide and lose their morphological and biochemical characteristics, whereas they maintain their phenotype for long periods of time when they are cultivated in alginate beads. Because cartilage has a low cellularity and is difficult to obtain in large quantities, the number of available cells often becomes a limiting factor in studies of chondrocyte biology. Therefore, we explored the possibility of restoring the differentiated properties of chondrocytes by cultivating them in alginate beads after two multiplication passages in monolayer. This resulted in the reexpression of the two main markers of differentiated chondrocytes: Aggrecan and type II collagen gene expression was strongly reinduced from day 4 after alginate inclusion and paralleled protein expression. However, 2 weeks were necessary for total suppression of type I and III collagen synthesis, indicators of a modulated phenotype. Interleukin-1beta, a cytokine that is present in the synovial fluid of rheumatoid arthritis patients, induces many metabolic changes on the chondrocyte biology. Compared with cells in primary culture, the production of nitric oxide and 92-kDa gelatinase in response to interleukin-1beta was impaired in cells at passage 2 in monolayer but was fully recovered after their culture in alginate beads for 2 weeks. This suggests that the effects of interleukin-1beta on cartilage depend on the differentiation state of chondrocytes. This makes the culture in alginate beads a relevant model for the study of chondrocyte biology in the presence of interleukin-1beta and other mediators of cartilage destruction in rheumatoid arthritis and osteoarthrosis.
Abstract: The understanding of cartilage disorders relies on the possibility of studying mechanisms which monitor the regulation of matrix protein genes through introducing efficiently and in a reproducible manner these genes, or their regulatory regions, into cells. To this end, we attempted to improve the transfection efficiency of rabbit articular chondrocytes by the calcium phosphate procedure. Transfection efficiencies were assessed by measuring the expression of the Lac Z reporter gene encoding β-galactosidase using anin situ staining (X-gal staining) and an enzymatic assay (β-galactosidase assay).Results revealed that addition of 4 U ml(-1) of hyaluronidase before and during transfection increases by 2 to 4-fold the transfection efficiency of rabbit articular chondrocytes. Furthermore, we demonstrated that the use of a "giant" calcium phosphate DNA coprecipitate gives a higher transfection efficiency and much more reproducible results than those obtained with classical small volumes of precipitates.
