Maria Balduzzi

Prostaglandin E2 synthesis is differentially affected by reactive nitrogen intermediates in cultured rat microglia and RAW 264.7 cells

We studied the effects of nitric oxide (NO) on prostanoid production, cyclooxygenase (COX‐2) expression and [3H]arachidonic acid (AA) release in RAW 264.7 macrophagic cells and rat microglial primary cultures. Inhibition of NO synthesis enhanced microglial prostanoid production without affecting that of RAW 264.7 cells. Both 3‐morpholinosydnonimine (SIN‐1), (which, by releasing NO and superoxide, leads to the formation of peroxynitrite), and S‐nitroso‐N‐acetylpenicillamine (SNAP), (which releases only NO), inhibited microglial prostanoid production, by preventing COX‐2 expression. In contrast, in RAW 264.7 cells, SIN‐1 enhanced both basal and LPS‐stimulated prostanoid production by upregulating COX‐2, while SNAP stimulated basal production and slightly inhibited the LPS‐induced production, as a cumulative result of enhanced AA release and depressed COX‐2 expression. Thus, reactive nitrogen intermediates can influence prostanoid production at distinct levels and in different way in the two cell types, and results obtained with RAW 264.7 cells can not be extrapolated to microglia.

Curcumin Protects against NMDA-Induced Toxicity: A Possible Role for NR2A Subunit

PURPOSE Curcumin, a phenolic compound extracted from the rhizome of Curcuma longa, was found to attenuate NMDA-induced excitotoxicity in primary retinal cultures. This study was conducted to further characterize curcumin neuroprotective ability and analyze its effects on NMDA receptor (NMDAr). METHODS NMDAr modifications were analyzed in primary retinal cell cultures using immunocytochemistry, whole-cell patch-clamp recording and western blot analysis. Cell death was evaluated with the TUNEL assay in primary retinal and hippocampal cultures. Optical fluorometric recordings with Fura 2-AM were used to monitor [Ca(2+)](i). RESULTS Curcumin dose- and time-dependently protected both retinal and hippocampal neurons against NMDA-induced cell death, confirming its anti-excitotoxic property. In primary retinal cultures, in line with the observed reduction of NMDA-induced [Ca(2+)](i) rise, whole-cell patch-clamp experiments showed that a higher percentage of retinal neurons responded to NMDA with low amplitude current after curcumin treatment. In parallel, curcumin induced an increase in NMDAr subunit type 2A (NR2A) level, with kinetics closely correlated to time-course of neuroprotection and decrease in [Ca(2+)](i). The relation between neuroprotection and NR2A level increase was also in line with the observation that curcumin neuroprotection required protein synthesis. Electrophysiology confirmed an increased activity of NR2A-containing NMDAr at the plasma membrane level. CONCLUSIONS These results confirm the neuroprotective activity of curcumin against NMDA toxicity, possibly related to an increased level of NR2A, and encourage further studies for a possible therapeutic use of curcumin based on neuromodulation of NMDArs.

The presence of astrocytes enhances beta amyloid-induced neurotoxicity in hippocampal cell cultures

A characteristic feature of neuritic plaques in Alzheimers disease is represented by the presence of activated astrocytes, surrounding dystrophic neurons and beta-amyloid deposition. To explore the role of astrocytes in in vitro beta-amyloid neurotoxicity, we studied the effect of beta-amyloid treatment in hippocampal neurons in two different cell models: pure cultures, where neurons were grown in absence of astrocytes and mixed cultures, where neurons were seeded on a confluent layer of astrocytes. We evaluated two characteristic aspects of in vitro beta-amyloid neurotoxicity: reduction of cell viability and degeneration of the neuritic tree. We demonstrated that neurons growing on astrocytes were more prone to the detrimental effect of the amyloid peptide, with respect to neurons grown in absence of the glial component. Our results support the hypothesis that beta-amyloid-astrocyte interaction can adversely condition neurons and contribute to neuronal damage in Alzheimers disease.

LIVER MITOCHONDRIA ALTERATIONS IN CHLOROFORM-TREATED SPRAGUE-DAWLEY RATS

The formation of a chloroform adduct produced by the reaction of the oxidative chloroform metabolite phosgene with two molecules of phosphatidylethanolamine has been previously demonstrated in liver mitochondria of phenobarbital-pretreated Sprague-Dawley (SD) rats. The aim of our study was to assess the influence of chloroform adduct mitochondrial accumulation on the hepatic mitochondria morphology. Liver mitochondrial ultrastructural alterations were analyzed by electron microscopy in SD rats administered with increasing doses of chloroform. Variation in the morphology of mitochondria, consisting of an increase of intertwined organelles, only rarely seen in control specimens, was observed at the lowest chloroform dose (180 mg/kg). At higher doses, mitochondrial damage progressed with swelling of the organelles and formation of megamitochondria. These megamitochondria were characterized by a dilution of the matrix, and often membranous whorls were found inside the matrix. The two distinct forms of cell death, necrosis and apoptosis, were first observed at 300 mg/kg of chloroform. Our results suggest that the formation and the accumulation of a chloroform-modified phosphatidylethanolamine in mitochondria induce ultrastructural modifications of these organelles. In conclusion, mitochondria are involved in chloroform-induced hepatotoxicity.

Modulation of the bystander effects induced by soluble factors in HaCaT cells by different exposure strategies.

Abstract The aim of this investigation was to explore whether the occurrence and the magnitude of radiation-induced, medium-mediated bystander effects could be influenced by the time of transfer of secreted bystander factors. HaCaT cells were exposed to 0.1 and 1.0 Gy of γ radiation. These doses did not induce a significant reduction in the clonogenic survival of irradiated cells compared to controls. Bystander cells either were co-cultured with irradiated cells or received medium from irradiated cells. The bystander effects analyzed included end points related to survival (clonogenic potential and cell proliferation) and DNA damage (micronucleus induction and γ-H2AX formation). The bystander effects we investigated either were lacking or varied from potentially protective to detrimental responses in relation to the dose of radiation and the time between irradiation of donor cells and bystander exposure. Our results suggest that the experimental time schedule is important for both the occurrence and the detection of bystander effects in vitro.

The contribution of human small intestine to chlorpyrifos biotransformation.

Despite the oral intake is the major route of exposure to chlorpyrifos for the general population, few data are available on human intestine biotransformation. In this study the contribution of chlorpyrifos (CPF) metabolism in human small intestine was investigated in microsomes from duodenum (HDM) and ileum/jejunum (HS2M) from 11 individual donors. Samples were characterized for testosterone hydroxylated metabolite formation and CYP content quantification by means of Western blotting. The two methods gave consistent results, evidencing the presence of CY3A4 and its-related activity in 10/11 samples, among which one showed also the presence of CYP2C9. Analogously, although with high interindividual variability (about 10 fold), CPF bioactivation to chlorpyrifos-oxon (CPFO) was observed in 10/11 HDM: intrinsic clearance highest value was 0.75 pmolCPFO/(mgproteinminμM). Detoxication to 3,5,6-trichloropyrin-2-ol formation was negligible. The comparison between HDM and HS2M indicates that most CPF bioactivation was confined in the duodenum, declining toward the distal ileum. Results suggest that following oral exposure, the small intestine CPF bioactivation, although much lower when compared to the total hepatic metabolism, could play a role in the pre-systemic CPF clearance, with CPFO transported into the lumen by the efflux P-glycoprotein and further metabolized by esterases.

Characterization of a Phospholipid Adduct Formed in Sprague Dawley Rats by Chloroform Metabolism: NMR Studies

The formation of a covalent adduct to a single phospholipid by the oxidative chloroform metabolite, phosgene, is demonstrated in liver mitochondria of phenobarbital‐pretreated Sprague Dawley (SD) rats treated with CHCl3. The densitometric analysis of the phosphorus stained extracted phospholipids showed that the formation of this adduct in liver mitochondria is accompanied by a decrease of phosphatidylethanolamine and cardiolipin. The characterization of this adduct was performed with a multinuclear NMR approach by comparison with the decreased phospholipids. Treatment of rats with [13C]chloroform resulted in an intense 13C NMR peak from either an esteric or amidic carbonyl. Very strong similarities in fatty acid composition were found between phosphatidylethanolamine and the phosgene‐modified PL, using 13C and 1H NMR spectroscopy. A multiplet at 3.91 ppm coupled to a signal at 3.41 ppm was shown by two‐dimensional 1H NMR in the adduct spectrum. This cross peak was interpreted as arising from the shifted resonances of the two PE head group methylene groups, due to the binding with phosgene. 31P spectrum of the adduct was identical to that of phosphatidylethanolamine. We concluded that the chloroform adduct is a modified phosphatidylethanolamine, with the phosgene‐derived carbonyl bound to the amine of the head group.

The complex interactions between radiation induced non-targeted effects and cancer

Radiation induced non-targeted effects have been widely investigated in the last two decades for their potential impact on low dose radiation risk. In this paper we will give an overview of the most relevant aspects related to these effects, starting from the definition of the low dose scenarios. We will underline the role of radiation quality, both in terms of mechanisms of interaction with the biological matter and for the importance of charged particles as powerful tools for low dose effects investigation. We will focus on cell communication, representing a common feature of non-targeted effects, giving also an overview of cancer models that have explicitly considered such effects.

Preliminary characterization of phospholipid adducts formed by [14C]-CHCl3 reactive intermediates in hepatocyte suspensions

The adducts produced in vitro by the reactive metabolites of [14C]-chloroform with total phospholipids (PLs) of freshly isolated hepatocytes have been characterized. The radical metabolite formed several adducts with all the major PL classes. These adducts seemed very likely to result from the unspecific attack of the radical on the PL fatty acyl chains. [14C]-Chloroform-derived phosgene caused the formation of a single PL adduct characterized by a ratio 14C:P of 1:4. This adduct was tentatively identified as an adduct of phosgene with two molecules of cardiolipin.

Glioblastoma stem cells: radiobiological response to ionising radiations of different qualities

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumour, with very poor prognosis. The high recurrence rate and failure of conventional treatments are expected to be related to the presence of radio-resistant cancer stem cells (CSCs) inside the tumour mass. CSCs can both self-renew and differentiate into the heterogeneous lineages of cancer cells. Recent evidence showed a higher effectiveness of C-ions and protons in inactivating CSCs, suggesting a potential advantage of Hadrontherapy compared with conventional radiotherapy for GBM treatment. To investigate the mechanisms involved in the molecular and cellular responses of CSCs to ionising radiations, two GBM stem cell (GSC) lines, named lines 1 and 83, which were derived from patients with different clinical outcomes and having different metabolic profiles (as shown by NMR spectroscopy), were irradiated with (137)Cs photons and with protons or C-ions of 62 MeV u(-1) in the dose range of 5-40 Gy. The biological effects investigated were: cell death, cell cycle progression, and DNA damage induction and repair. Preliminary results show a different response to ionising radiation between the two GSC lines for the different end points investigated. Further experiments are in progress to consolidate the data and to get more insights on the influence of radiation quality.