IDO, COX and iNOS have an important role in the proliferation of Neospora caninum in neuron/glia co-cultures.

Central nervous system (CNS) is the main site for encystment of Neospora caninum in different animal species. In this tissue, glial cells (astrocytes and microglia) modulate responses to aggression in order to preserve homeostasis and neuronal function. Previous data showed that when primary cultures of glial cells are infected with N. caninum, they develop gliosis and the immune response is characterized by the release of TNF and IL-10, followed by the control of parasite proliferation. In order to elucidate this control, three enzymatic systems involved in parasite-versus-host interactions were observed on a model of neuron/glia co/cultures obtained from rat brains. Indoleamine 2,3-dioxygenase (IDO), induced nitric oxide synthase (iNOS) responsible for the catabolism of tryptophan and arginine, respectively, and cycloxigenase (COX) were studied comparing their modulation by respective inhibitors with the number of tachyzoites or the immune response measured by the release of IL-10 and TNF. Cells were treated with the inhibitors of iNOS (1.5 mM L-NAME), IDO (1 mM 1-methyl tryptophan), COX-1 (1 µM indomethacin) and COX-2 (1 µM nimesulide) before infection with tachyzoites of N. caninum (1:1 cell: parasite). After 72 h of infection, immunocytochemistry showed astrogliosis and a significant increase in the number and length of neurites, compared with uninfected co-cultures, while an increase of IL-10 and TNF was verified. N. caninum did not change iNOS activity, but the inhibition of the basal levels of this enzyme stimulated parasite proliferation. Additionally, a significant increase of about 40% was verified in the IDO activity, whose inhibition caused 1.2-fold increase in parasitic growth. For COX-2 activity, infection of cultures stimulated a significant increase in release of PGE2 and its inhibition by nimesulide allowed the parasitic growth. These data indicate that iNOS, IDO and COX-2 control the proliferation of N. caninum in this in vitro model. On the other hand, the release of IL-10 by glia besides modulating the inflammation also allow the continuity of parasitism.

Effects of IFN-γ, TNF-α, IL-10 and TGF-ß on Neospora caninum infection in rat glial cells.

Neospora caninum causes abortion in cattle and neurological disorders in dogs. The immunological response to this parasite has been described as predominantly of the Th1 type. However, infected primary glial cell cultures release IL-10 and IL-6 but not IFN-γ. This suggests a rather protective response of the glia to avoid inflammatory damage of the nervous tissue. In this study, we investigated the effects of pro-inflammatory cytokines in primary mixed cultures of rat astrocytes and microglia infected with N. caninum. The cells were treated with either IFN-γ, TNF-α, anti-IL-10 or anti-TGF-ß antibodies and were infected with parasite tachyzoites 24h later. Trypan Blue exclusion and MTT assays were performed to test cell viability. It was observed that cytokines, antibody treatment and in vitro infection did not reveal significant cell death in the various culture conditions. Treatment with 50, 150 and 300 IU/mL of either IFN-γ or TNF-α reduced tachyzoites numbers in cultures by 36.7%, 54.8% and 63.8% for IFN-γ and by 27.6%, 38.4% and 29.7% for TNF-α, respectively. In the absence of IL-10 and TGF-ß, tachyzoite numbers were reduced by 52.8% and 41.5%, respectively. While IFN-γ (150 and 300 IU/mL) increased the nitrite levels in uninfected cells, parasite infection seemed to reduce the nitrite levels, and this reduction was more expressive in IFN-γ-infected cells, thereby suggesting an inhibitory effect on its production. However, TNF-α, IL-10 and TGF-ß did not affect the nitrite levels. Basal PGE(2) levels also increased by 17% and 25%; 78% and 13% in uninfected and infected cells treated with IFN-γ or anti-TGF-ß, respectively. Nevertheless, the antibody neutralization of IL-10 reduced PGE(2) release significantly. These results highlight the possibility of a combined effect between the IFN-γ and parasite evasion strategies and show that the IFN-γ, TNF-α, IL-10 and TGF-ß cytokines participate in parasite proliferation control mechanisms.

Antiproliferative, proapoptotic and morphogenic effects of the flavonoid rutin on human glioblastoma cells.

In this study, we investigated the effects of the flavonoid rutin (3,3',4',5,7-pentahydroxyflavone-3-rutinoside) on glioma cells, using the highly proliferative human cell line GL-15 as a model. We observed that rutin (50-100µM) reduced proliferation and viability of GL-15 cells, leading to decreased levels of ERK1/2 phosphorylation (P-ERK1/2) and accumulation of cells in the G2 phase of the cell cycle. On the other hand, 87.4% of GL-15 cells exposed to 100µM rutin entered apoptosis, as revealed by flow cytometry after AnnexinV/PI staining. Nuclear condensation and DNA fragmentation were also observed, further confirming that apoptosis had occurred. Moreover, the remaining cells that were treated with 50µM rutin presented a morphological pattern of astroglial differentiation in culture, characterised by a condensed cell body and thin processes with overexpression of GFAP. Because of its capacity to induce differentiation and apoptosis in cultured human glioblastoma cells, rutin could be considered as a potential candidate for malignant gliomas treatment.

Catechol cytotoxicity in vitro: induction of glioblastoma cell death by apoptosis.

The exposure to benzene is a public health problem. Although the most well-known effect of benzene is hematopoietic toxicity, there is little information about the benzene and its metabolites effects on the central nervous system (CNS). This study examined the toxic effects of 1,2-dihydroxybenzene (catechol), a benzene metabolite, to human glioblastoma GL-15 cells. GL-15 cell cultures were used as a model to provide more information about the toxic effects of aromatic compounds to the CNS. Catechol induced time- and concentration-dependent cytotoxic effects. Morphological changes, such as the retraction of the cytoplasm and chromatin clumping, were seen in cells exposed to 200 microM catechol for 48 hours. In cells exposed to 600 microM catechol for 48 hours, 78.0% of them presented condensed nuclei, and the Comet assay showed DNA damage. The percentage of cells labeled with annexin V (apoptotic cells) was greater in the group exposed to catechol (20.7%) than in control cells (0.4%). Exposure to catechol at concentrations greater than 100 microM enhanced Bax levels, and a decrease in Bcl-2 level was observed after the exposure to 600 microM catechol for 48 hours. Furthermore, catechol depleted reduced glutathione. Hence, catechol induced cell death mainly by apoptosis.

Neospora caninum: early immune response of rat mixed glial cultures after tachyzoites infection.

Neospora caninum causes neurologic disease in dogs and abortion in cattle. Little is known about the immune response of the CNS against this protozoan. The aim of this study was to evaluate production of IL-6, IL-10, TNF-alpha, IFN-gamma, and NO in rat mixed glial cell cultures infected by N. caninum. IFN-gamma was not observed. The mean cytokine released after 24 and 72 h of infection were 3.8+/-0.6 and 3.7+/-0.6 pg TNF-alpha/mg protein and 2.7+/-0.69 and 4.1+/-0.64 pg IL-10/mg protein, respectively, and more than 8.0 pg IL-6/mg protein for both time points. NO levels increased 24h post-infection (2.3+/-0.8 pg/mg protein) until 72 h (4.2+/-1.1 pg/mg protein) and the number of tachyzoites reduced with the time. Our results show high levels of regulatory cytokines that may suppress the harmful effects of IFN-gamma; high levels of TNF-alpha and NO may represent an effective response by infected glial cells against N. caninum.

Genotoxicity and morphological changes induced by the alkaloid monocrotaline, extracted from Crotalaria retusa, in a model of glial cells.

Plants of Crotalaria genus (Leguminosae) present large amounts of the pyrrolizidine alkaloid monocrotaline (MCT) and cause intoxication to animals and humans. Therefore, we investigated the MCT-induced cytotoxicity, morphological changes, and oxidative and genotoxic damages to glial cells, using the human glioblastoma cell line GL-15 as a model. The comet test showed that 24h exposure to 1-500microM MCT and 500microM dehydromonocrotaline (DHMC) caused significant increases in cell DNA damage index, which reached 42-64% and 53%, respectively. Cells exposed to 100-500microM MCT also featured a contracted cytoplasm presenting thin cellular processes and vimentin destabilisation. Conversely, exposure of GL-15 cells to low concentrations of MCT (1-10microM) clearly induced megalocytosis. Moreover, MCT also induced down regulation of MAPs, especially at the lower concentrations adopted (1-10microM). Apoptosis was also evidenced in cells treated with 100-500microM MCT, and a later cytotoxicity was only observed after 6 days of exposure to 500microM MCT. The data obtained provide support for heterogenic and multipotential effects of MCT on GL-15 cells, either interfering on cell growth and cytoskeletal protein expression, or inducing DNA damage and apoptosis and suggest that the response of glial cells to this alkaloid might be related to the neurological signs observed after Crotalaria intoxication.

Monocrotaline pyrrol is cytotoxic and alters the patterns of GFAP expression on astrocyte primary cultures.

Dehydromonocrotaline (DHMC) is the main monocrotaline active cytochrome P450's metabolite, and has already been assessed in the CNS of experimentally intoxicated rats. DHMC effects were here investigated toward rat astroglial primary cultures regarding cytotoxicity, morphological changes and regulation of GFAP expression. Cells, grown in DMEM supplemented medium, were treated with 0.1-500 microM DHMC, during 24- and 72-h. According to MTT and LDH tests, DHMC was toxic to astrocytes after 24-h exposure at 1 microM, and induced membrane damages at 500 microM. Rosenfeld dying showed hypertrophic astrocytes after 72-h exposure to 0.1-1 microM DHMC. GFAP immunocytochemistry and western immunoblot revealed an increase of GFAP labelling and expression, suggesting an astrogliotic reaction to low concentrations of DHMC. At higher concentrations (10-500 microM), astrocytes shrank their bodies and retracted their processes, presenting a more polygonal phenotype and a weaker expression on GFAP labelling Nuclear chromatin staining by Hoechst-33258 dye, revealed condensed and fragmented chromatin in an important proportion (+/-30%) of the astrocytes exposed to 100-500 microM DHMC, suggesting signs of apoptosis. Our results confirm a cytotoxic and dose-dependent effect of DHMC on cultures of rat cortical astrocytes, leading to apoptotic figures. These effects might be related to the neurological damages and clinical signs observed in animals intoxicated by Crotalaria.

The flavonoid rutin induces astrocyte and microglia activation and regulates TNF-alpha and NO release in primary glial cell cultures.

Astrocyte and microglia cells play an important role in the central nervous system (CNS). They react to various external aggressions by becoming reactive and releasing neurotrophic and/or neurotoxic factors. Rutin is a flavonoid found in many plants and has been shown to have some biological activities, but its direct effects on cells of the CNS have not been well studied. To investigate its potential effects on CNS glial cells, we used both astrocyte primary cultures and astrocyte/microglia mixed primary cell cultures derived from newborn rat cortical brain. The cultures were treated for 24 h with rutin (50 or 100 micromol/L) or vehicle (0.5% dimethyl sulfoxide). Mitochondrial function on glial cells was not evidenced by the MTT test. However, an increased lactate dehydrogenase activity was detected in the culture medium of both culture systems when treated with 100 micromol/L rutin, suggesting loss of cell membrane integrity. Astrocytes exposed to 50 micromol/L rutin became reactive as revealed by glial fibrillary acidic protein (GFAP) overexpression and showed a star-like phenotype revealed by Rosenfeld's staining. The number of activated microglia expressing OX-42 increased in the presence of rutin. A significant increase of nitric oxide (NO) was observed only in mixed cultures exposed to 100 micromol/L rutin. Enhanced TNFalpha release was observed in astrocyte primary cultures treated with 100 micromol/L rutin and in mixed primary cultures treated with 50 and 100 micromol/L, suggesting different sensitivity of both activated cell types. These results demonstrated that rutin affects astrocytes and microglial cells in culture and has the capacity to induce NO and TNFalpha production in these cells. Hence, the impact of these effects on neurons in vitro and in vivo needs to be studied.

Alkaloids from Prosopis juliflora leaves induce glial activation, cytotoxicity and stimulate NO production.

Prosopis juliflora is used for feeding cattle and humans. Intoxication with the plant has been reported, and is characterized by neuromuscular alterations and gliosis. Total alkaloidal extract (TAE) was obtained using acid/basic-modified extraction and was fractionated. TAE and seven alkaloidal fractions, at concentrations ranging 0.03-30 microg/ml, were tested for 24h on astrocyte primary cultures derived from the cortex of newborn Wistar rats. The MTT test and the measure of LDH activity on the culture medium, revealed that TAE and fractions F29/30, F31/33, F32 and F34/35 were cytotoxic to astrocytes. The EC(50) values for the most toxic compounds, TAE, F31/33 and F32 were 2.87 2.82 and 3.01 microg/ml, respectively. Morphological changes and glial cells activation were investigated through Rosenfeld's staining, by immunocytochemistry for the protein OX-42, specific of activated microglia, by immunocytochemistry and western immunoblot for GFAP, the marker of reactive and mature astrocytes, and by the production of nitric oxide (NO). We observed that astrocytes exposed to 3 microg/ml TAE, F29/30 or F31/33 developed compact cell body with many processes overexpressing GFAP. Treatment with 30 microg/ml TAE and fractions, induced cytotoxicity characterized by a strong cell body contraction, very thin and long processes and condensed chromatin. We also observed that when compared with the control (+/-1.34%), the proportion of OX-42 positive cells was increased in cultures treated with 30 microg/ml TAE or F29/30, F31/33, F32 and F34/35, with values raging from 7.27% to 28.74%. Moreover, incubation with 3 microg/ml F32, 30 microg/ml TAE, F29/30, F31/33 or F34/35 induced accumulation of nitrite in culture medium indicating induction of NO production. Taken together these results show that TAE and fractionated alkaloids from P. juliflora act directly on glial cells, inducing activation and/or cytotoxicity, stimulating NO production, and may have an impact on neuronal damages observed on intoxicated animals.

Different effects of arborinine alkaloid obtained from Brazilian Erthela baihensis on spleen and thymus cells stimulated in vitro with different mitogens.

The present study has examined the effects of arborinine, an alkaloid obtained from Erthela bahiensis, a Brazilian plant popularly used as diuretic, antidiabetic, antithermic and expectorant, on the viability and function of immune system cells in vitro using a murine model. Rat spleen and thymus cells were cultured with 10 nM, 1 microM, 10 microM of the drug in the presence or absence of pokeweed (PWM), lipopolysaccharide (LPS), or concanavallin (ConA) mitogens. Cellular proliferation was analyzed by H3-thymidin uptake after 48 and 72 hr. Our results showed an inhibitory effect of arborinine on splenocytes proliferation under ConA or PWM stimulation and increased apoptosis on splenocytes and thymocytes stimulated with PWM in 24 hr. A decrease was observed on Interferon gamma (IFN-gamma) production by ConA- or LPS-stimulated splenocytes in 48 hr and 72 hr and ConA- or PWM-stimulated thymocytes in 72 hr. In contrast, an increase on lymphoproliferation was observed on LPS-stimulated splenocytes and ConA- or PWM-stimulated thymocytes in 48 hr. On this period, apoptosis decreased on LPS- or PWM-stimulated splenocytes and IFN-gamma production increased in PWM stimulated thymocytes. Arborinine also induced a decrease on Interleukin-10 production by splenocytes and thymocytes stimulated with ConA or PWM. There was no significant variation on the necrosis rate of the cells treated with arborinine or any change on their viability or function values in the absence of mitogenic stimulus.

Astroglial cells in primary culture: a valid model to study Neospora caninum infection in the CNS.

The protozoan Neospora caninum has a veterinary importance because it causes abortion in cattle and neuromuscular alterations in dogs. We infected rat astrocytes, in vitro, with different concentrations of N. caninum. Astrocytes responded to infection by producing the pro-inflammatory cytokine TNF-alpha and the neurotoxic-free radical NO, 24 and 72 h post-infection. These data suggest that astrocytes, which are essential for brain function, are targets for the parasite and this represents a practical and valid model to study the effects of N. caninum on the CNS.

Neospora caninum: infection induced IL-10 overexpression in rat astrocytes in vitro.

The effect of Neospora caninum, a parasite that causes abortion and neuromuscular changes, has been investigated on a major population of neural cells, the astrocytes. Highly enriched astroglial primary cultures obtained from neonatal rats were infected after 21 days of culture. Astroglial reactivity, IL-10 and IFN-gamma expression, and cell viability (lactate dehydrogenase activity, metabolization of tetrazolium salt, and trypan blue exclusion assay) have been investigated after 24 and 72 h of infection. Astroglial hypertrophy, gliofilament reorganization, metabolic changes suggesting hypoxia and a strong IL-10 release have been observed in the infected cells. These results show that neural cells are targets for the parasite and that astrocytes may contribute to the CNS immune response to the parasite.

Toxic effects of apomorphine on rat cultured neurons and glial C6 cells, and protection with antioxidants.

Many catechol derivatives are currently used as drugs, even if they produce reactive oxygen species that may cause tissue damage. Among them, apomorphine, a potent dopamine agonist, displays efficient anti-parkinsonian properties, but the consequences of its oxidant and toxic properties have been poorly investigated on in vitro models. In the present work, we investigated apomorphine cytotoxicity by incubating cultures of rat glioma C6 cells and primary cultures of neurons with different concentrations of the drug. Apomorphine-promoted cell death was proportional to its concentration and was time-dependent. The ED(50) of apomorphine on C6 cell death after 48 hr was about 200 microM. The cytotoxic effects induced by apomorphine were correlated to its autoxidation, which leads to the formation of reactive oxygen species, semiquinones, quinones, and a melanin-like pigment. C6 cells that underwent treatment with 400 microM apomorphine for 6 hr displayed features of necrosis, including loss of membrane integrity, degeneration of mitochondria, and DNA fragmentation. Thiols, such as cysteine, N-acetyl-L-cysteine, and glutathione, significantly protected cultured neurons and C6 cells against apomorphine-induced cytotoxicity. Thiols also inhibited apomorphine autoxidation. These data strongly suggest that apomorphine cytotoxicity towards neurons and C6 cells results from an intracellular oxidative stress.

Inhibition of rat brain microsomal cytochrome P450-dependent dealkylation activities by an oxidative stress.

There is increasing evidence that an oxidative stress not only alters cellular lipids and nucleic acids, but also numerous proteins. This oxidation results in alterations of some cellular functions, either by reversible modifications allowing a post-transcriptional regulation of enzyme activities or receptor affinities, or by irreversible modifications of the protein, triggering its inactivation and destruction. In the present work, we examined the effects of an experimental oxidative stress on rat brain microsomal cytochrome P450-dependent dealkylation activities. For that purpose, superoxide anions were produced either by the NADPH-dependent redox cycling of a quinine, menadione, or by the addition of apomorphine, which produces by autoxidation both superoxide anions and apomorphine-derived quinones. The inhibition of brain cytochrome P450-dependent alkoxyresorufin O-dealkylase activities was dependent on both menadione or apomorphine concentrations. Simultaneously, an increase of microsomal carbonyl groups was recorded. Immunoblotting characterization of brain microsomal oxidized protein was carried out, using antibodies raised against 2,4-dinitrophenylhydrazine as a reagent of protein carbonyl groups, and a revelation by a chemiluminescence method. We observed an increase in cerebral CYP1A protein oxidation, related to menadione concentration, suggesting that oxidation of cytochrome P450 protein may result in its catalytic inactivation.

Glucuronidation of apomorphine.

Apomorphine, a dopaminergic receptor agonist, is largely used in the therapy of Parkinson's disease. In this study, we characterized the glucuronidation of apomorphine and other catechols in rat liver and brain microsomes, using UDP-[U-14C]glucuronic acid and separation of the glucuronides formed by a thin layer chromatographic method. rat liver microsomes glucuronidate apomorphine at a significant rate, that was increased in the presence of dithiothreitol. Two apomorphine glucuronides were separated by high pressure liquid chromatography. We showed by electrospray mass spectrometry that both products were monoglucuronides. Other catechols were also glucuronidated in liver microsomes at various rates, and among them, 4-nitrocatechol was the most efficiently conjugated. in rat brain microsomes, only 4-nitrocatechol was significantly glucuronidated, suggesting that in the liver, several uridine-diphosphate glucuronosyltransferase (UGT) isoforms participate to the conjugation of catechols. To determine which isoforms catalyze apomorphine glucuronidation, two recombinant enzymes expressed in V79 cells were used. The isoform UGT1A6 was unable to glucuronidate apomorphine, but we observed a significant activity catalyzed by the isoform UGT2B1. These results provide, to our knowledge, the first demonstration of apomorphine conjugation by recombinant UGT2B1, and the first evidence of the lack of apomorphine glucuronidation in the rat brain.

Mechanisms of apomorphine cytoxicity towards rat glioma C6 cells: protection by bovine serum albumin and formation of apomorphine-protein conjugates.

Apomorphine cytotoxicity towards rat glioma C6 cells was recently demonstrated to be time- and concentration-dependent. In the present work, the mechanism of cytotoxicity of apomorphine was further studied in the C6 cell line. We showed that bovine serum albumin partially protects C6 cells against apomorphine cytotoxicity. However, serum albumin did not prevent apomorphine autoxidation and melanin formation, suggesting that this protein scavenges apomorphine reactive products formed during its oxidation. The use of radioactive tracers, fluorimetry and protein electrophoresis showed that apomorphine autoxidation products covalently and nonspecifically bind to serum albumin and to rat liver microsomes. L-Cysteine, which is a thiol reagent that inhibits apomorphine autoxidation also prevented the formation of apomorphine-serum albumin adducts. These results suggest that quinone derivatives formation and oxidative stress should be responsible for apomorphine cytotoxicity.

Drug metabolizing enzymes in cerebrovascular endothelial cells afford a metabolic protection to the brain.

The brain is partially protected from chemical insults by a physical barrier mainly formed by the cerebral microvasculature, which prevents penetration of hydrophilic molecules in the cerebral extracellular space. This results from the presence of tight junctions joining endothelial cells, and from a low transcytotic activity in endothelial cells, inducing selective permeability properties of cerebral microvessels that characterize the blood-brain barrier. The endothelial cells provide also, as a result of their drug-metabolizing enzymes activities, a metabolic barrier against potentially penetrating lipophilic substances. It has been established that in cerebrovascular endothelial cells, several families of enzymes metabolize potentially toxic lipophilic substrates from both endogenous and exogenous origin to polar metabolites, which may not be able to penetrate further across the blood-brain barrier. Enzymes of drug metabolism present at brain interfaces devoid of blood-brain barrier, like circumventricular organs, pineal gland, and hypophysis, that are potential sites of entry for xenobiotics, display higher activities than in cerebrovascular endothelial cells, and conjugation activities are very high in the choroid plexus. Finally, xenobiotic metabolism normally results in detoxication, but also in some cases in the formation of pharmacologically active or neurotoxic products, possibly altering some blood-brain barrier properties.

Dietary Antioxidant Deficiency Facilitates Cortical Spreading Depression Induced by Photoactivated Riboflavin.

It is known that the photoactivation of riboflavin produces superoxide radicals. We investigated the ability of this process to elicit spreading depression (SD) in the cerebral cortex of adult rats receiving either a normal diet (control group; n = 9) or fed a diet free from vitamins C and E during 4-6 weeks prior to the experiment (deficient group; n = 15). SD was initially elicited, at 20 min intervals, by 2% KC1 topically applied for 1 min to a point (2-3 mm in diameter) on the dura mater at the frontal cortex and SD propagation was monitored by both EEG and DC-recordings at two points of the parietal region. After a 1-2 h "baseline" recording of KCl-elicited SD, tests were performed with 1.0 mM riboflavin applied to the same frontal region and illuminated by a white light bulb (40 W, 10-15 cm from the cortical surface, for 1-3 min). In the control group, 37 applications of riboflavin + light were performed (average: 4.1 applications per rat; range: 3-7) and 11 of these applications (29.7%) elicited SD in 7 out of the 9 rats. In the deficient group, the effectiveness of photoactivated riboflavin to elicit SD increased significantly to 62.8% (44 out of 70 applications; 15 out of 15 rats; average: 4.7 applications per rat; range: 3-6; P < 0.05). Elicitation of SD was not obtained, either by illumination of an equivalent volume of Ringer solution applied to the same region, or by riboflavin applied without illumination. The results demonstrate that photoactivated riboflavin is capable of eliciting SD in the rat cerebral cortex, and that dietary deficiency of the antioxidant vitamins C and E can enhance brain susceptibility to this process.