Exposing primary rat retina cell cultures to γ-rays: An in vitro model for evaluating radiation responses.

Retinal tissue can receive incidental γ-rays exposure during radiotherapy either of tumors of the eye and optic nerve or of head-and-neck tumors, and during medical diagnostic procedures. Healthy retina is therefore at risk of suffering radiation-related side effects and the knowledge of pathophysiological response of retinal cells to ionizing radiations could be useful to design possible strategies of prevention and management of radiotoxicity. In this study, we have exploited an in vitro model (primary rat retinal cell culture) to study an array of biological effects induced on retinal neurons by γ-rays. Most of the different cell types present in retinal tissue - either of the neuronal or glial lineages - are preserved in primary rat retinal cultures. Similar to the retina in situ, neuronal cells undergo in vitro a maturational development shown by the formation of polarized neuritic trees and operating synapses. Since 2 Gy is the incidental dose received by the healthy retina per fraction when the standard treatment is delivered to the brain, retina cell cultures have been exposed to 1 or 2 Gy of γ-rays at different level of neuronal differentiation in vitro: days in vitro (DIV)2 or DIV8. At DIV9, retinal cultures were analyzed in terms of viability, apoptosis and characterized by immunocytochemistry to identify alterations in neuronal differentiation. After irradiation at DIV2, MTT assay revealed an evident loss of cell viability and ßIII-tubulin immunostaining highlighted a marked neuritic damage, indicating that survived neurons showed an impaired differentiation. Differentiated cultures (DIV8) appeared to be more resistant with respect to undifferentiated, DIV2 cultures, both in terms of cell viability and differentiation. Apoptosis evaluated with TUNEL assay showed that irradiation at both DIV2 and DIV8 induced a significant increase in the apoptotic rate. To further investigate the effects of γ-rays on retinal neurons, we evaluated the expression of synaptic proteins, such as SNAP25 and synaptophysin. WB and immunofluorescence analysis showed an altered expression of these proteins in particular when cultures were irradiated at DIV2. To evaluate the effect of γ-rays on photoreceptors, we studied the expression of rhodopsin in WB analysis and immunofluorescence. Our results confirm data from the literature that differentiated photoreceptors appear to be more resistant to irradiation respect to other retinal cell types present in cultures. The results obtained suggest that γ-rays exposure of primary retinal cultures may contribute to shed further light on the mechanisms involved in γ-radiation-induced neurodegeneration.

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.

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.

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.

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

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 gamma 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 gamma-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.

Astrocyte modulation of in vitro beta-amyloid neurotoxicity.

In Alzheimer's disease brain, beta-amyloid (Abeta) deposition is accompanied by astrocyte activation, whose role in the pathogenesis of the disease is still unclear. To explore the subject, we compared Abeta neurotoxicity in pure hippocampal cultures and neuronal-astrocytic cocultures, where astrocytes conditioned neurons but were not in contact with them or Abeta. In the presence of astrocytes, neurons were protected from Abeta neurotoxicity. Neuritic dystrophy was reduced, synapses were partially preserved, and apoptosis was contrasted. The protection disappeared when astrocytes were also treated with Abeta, suggesting that Abeta-astrocyte interaction is deleterious for neurons. This was supported by comparing Abeta neurotoxicity in pure neurons and neurons grown on astrocytes. In this case, where astrocytes were also in contact with Abeta, neuritic damage was enhanced and expression of synaptic vesicle proteins decreased. Our results suggest that astrocytes can protect neurons from Abeta neurotoxicity, but when they interact with Abeta, the protection is undermined and neurotoxicity enhanced.

Environmental fine particulate matter (PM 2.5) activates the RAW 264.7 macrophage cell line even at very low concentrations as revealed by 1H NMR.

Because of the association between inhalation of airborne particulate matter (PM) and human respiratory and cardiovascular disease, it is necessary to understand the tissue damage induced by these particles. One of the cell types principally involved in the body's reaction to PM are macrophages, which remove particles in the airway passages and the lungs through phagocytosis. In fact, when macrophages are exposed to a toxic agent such as PM, they undergo a series of changes (including variations in morphology, an increase in glycolysis, and consequent lactate production and the release of cytokines such as interleukin-6 and tumor necrosis factor-alpha) necessary to transform them from "resting" to "activated" macrophages. Because (1)H NMR is extremely useful in monitoring, noninvasively, macrophage metabolism and because this technique has never been utilized to examine macrophage activation after exposure to PM, it was the purpose of the present study to investigate the effects of PM exposure on the RAW 264.7 stabilized macrophage cell line using (1)H NMR spectroscopy. PM with a diameter <2.5 microm (PM 2.5) was utilized because a closer association to mortality and adverse respiratory health effects has been found with this fraction than with particles of a larger size. Measurements were conducted on whole cells at both 500 and 700 MHz as well as on perchloric acid extracts at 700 MHz. Significant variations in numerous metabolites were seen at very low concentrations of PM 2.5. Many of these changes point to activation of RAW 264.7 macrophages even at doses of PM 2.5 much lower than those commonly employed in cell studies. These results are particularly significant since the same concentrations of PM did not induce changes in morphology and release of cytokines in these cells. Therefore, (1)H NMR spectroscopy is an extremely sensitive probe in observing subtle variations in macrophages after exposure to PM 2.5.

[Biological effects of PM10 relevant to human health]. / Effetti biologici del PM10 rilevanti per la salute dell'uomo.

Recent epidemiological studies have shown a consistent association between ambient levels of inhalable particles (PM10) and exacerbation of respiratory diseases as well as cardiovascular morbidity and adult mortality in high risk groups. The particles responsible of the observed health effects are unknown; it seems that different particles could be related to different effects, depending on the deposition pattern in the airways and on the chemical reactivity. Larger particles could be more related to upper airway and tracheobronchial effects while the smaller carbonaceous particles seem to be preferentially involved in inflammation and cardiopulmonary injury. It has been proposed that the possible biological mechanisms of action of PM could be related to its radical activity and the induction of oxidative stress and lung inflammation followed by systemic low grade inflammation and pro-coagulant state.

Differences in the biological activity of two PM3.3 components: carbonaceous and silica particles.

In this study we compared the biological reactivity of PM3.3 with those of carbon black (CB) and respirable silica particles, monitored by in vitro hemolytic potential and morphological alterations, in order to evaluate the correlations between the different physico-chemical characteristics of the three types of particulate and their biological effects. Carbon black and silica particles were used as reference environmental particles in order to limit the number of the urban PM variables, which is a mixture highly heterogeneous. Our data suggest that the urban PM3.3 have a similar surface reactivity as CB. In fact, when the percent of hemolysis were plotted against particle surface per volume units, the PM3.3 activity did not differ significantly from that of CB. This observation is in agreement with the SEM morphological evaluations of treated erythrocytes because the more abundant alteration in PM3.3-treated cells was the stomatocytic transformation (main feature of CB-treated red blood cells), followed by echinocytic transformation (observed in silica-treated cells).

Modulation of PGE(2) and TNFalpha by nitric oxide and LPS-activated RAW 264.7 cells.

Prostaglandins (PGs), the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and the cytokine TNFalpha play major roles in inflammation and they are synthesised mainly by macrophages. Their syntheses have been shown to be regulated by several factors, including nitric oxide, a further important macrophage product. Since both positive and negative regulations of PGs and TNFalpha synthesis by NO have been reported, we sought to understand the mechanisms underlying these opposite NO effects by using a recent class of NO releasing compounds, the NONOates, which have been shown to release NO in a controlled fashion. To this aim, we analysed the effect of NO released from PAPA/NO (t1/2 15 min) and DETA/NO (t1/2 20 h) in RAW 264.7 cells. Both NONOates were used at the same concentrations allowing the cell cultures to be exposed either at high levels of NO for brief time (PAPA/NO) or at low levels of NO for long time (DETA/NO). We found that the two NONOates had opposite effect on basal TNFalpha release, being increased by PAPA/NO and decreased by DETA/NO, while they did not affect the release stimulated by LPS. At variance, both NONOates increased the basal PGE(2) production, while the LPS-stimulated production was slightly increased only by PAPA/NO. The modulation of PGE(2) synthesis was the result of the distinct effects of the two NO-donors on either arachidonic acid (AA) release or cyclooxygense-2 (COX-2) expression, the precursor and synthetic enzyme of PGs, respectively. Indeed, in resting cultures AA release was enhanced only by PAPA/NO whereas COX-2 expression was moderately upregulated by both donors. In LPS activated cells, both NONOates induced AA release, although with different kinetics and potencies, but only DETA/NO significantly increased COX-2 expression. In conclusion, by comparing the activities of these two NONOates, our observations indicate that level and time of exposure to NO are both crucial in determining the molecular target and the final result of the interactions between NO and inflammatory molecules.