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

Toxicon 55 (2010) 105–117

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Genotoxicity and morphological changes induced by the alkaloid monocrotaline, extracted from Crotalaria retusa, in a model of glial cells J.P. Silva-Neto a, R.A. Barreto a, B.P.S. Pitanga a, C.S. Souza a, V.D. Silva a, A.R. Silva a, E.S. Velozo b, S.D. Cunha c, M.J.M. Batatinha d, M. Tardy e, C.S.O. Ribeiro a, M.F.D. Costa a, R.S. El-Bacha´ a, S.L. Costa a, * a

´ rio de Neuroquı´mica e Biologia Celular, Instituto de Cieˆncias da Sau ´ de, Departamento de Biofunça ˜o/Bioquı´mica, Salvador, BA 40.110-100, Brazil Laborato ´cia, Universidade Federal da Bahia, Salvador, BA 40170-290, Brazil ´rio de Pesquisa em Mate´ria Me´dica, Faculdade de Farma Laborato c ˆnica, Instituto de Quı´mica, Universidade Federal da Bahia, Salvador, BA 40.170-290, Brazil Departamento de Quı´mica Orga d ´ria, Departamento de Patologia e Clı´nica, Universidade Federal da Bahia, Salvador, BA 40.110-170, Brazil ´rio de Toxicologia, Escola de Medicina Veterina Laborato e Universite´ Paris XII Val de Marne Faculte´ de Me´dicine, Rue du Ge´neral Sarrail, 94.010, Val-de-Marne, Cre´teil, Cedex, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 February 2009 Received in revised form 8 July 2009 Accepted 9 July 2009 Available online 15 July 2009

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 24 h exposure to 1–500 mM MCT and 500 mM dehydromonocrotaline (DHMC) caused significant increases in cell DNA damage index, which reached 42–64% and 53%, respectively. Cells exposed to 100–500 mM MCT also featured a contracted cytoplasm presenting thin cellular processes and vimentin destabilisation. Conversely, exposure of GL-15 cells to low concentrations of MCT (1–10 mM) clearly induced megalocytosis. Moreover, MCT also induced down regulation of MAPs, especially at the lower concentrations adopted (1–10 mM). Apoptosis was also evidenced in cells treated with 100–500 mM MCT, and a later cytotoxicity was only observed after 6 days of exposure to 500 mM 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. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Monocrotaline Pyrrolizidine alkaloids Glia Genotoxicity Vimentin MAPs

1. Introduction Crotalaria retusa is a plant highly toxic to many animal species and to humans (Rose et al., 1957; Gardiner et al., 1965; Curran et al., 1996; Hooper and Scanlan, 1977; Alfonso et al., 1993; Nobre et al., 2004). Its toxicity has been

* Corresponding author. Laborato´rio de Neuroquı´mica e Biologia Celular, Departamento de Biofunça˜o Universidade Federal da Bahia Av. Reitor Miguel Calmon S/N, Canela, Salvador, BA 40.110-100, Brazil. Tel.: þ55 71 32838919; fax: þ55 71 32838884. E-mail addresses: [email protected], [email protected] (S.L. Costa). 0041-0101/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2009.07.007

attributed to the pyrrolizidine alkaloid (PA) monocrotaline (MCT), which is found abundantly in its seeds. It is well known that this alkaloid may be activated by hepatic cytochrome P450 complex (CYP450), which generates active metabolites such as dehydromonocrotaline (DHMC). Although MCT is primarily considered a hepatotoxic alkaloid, animals more susceptible to its intoxication, such as horses, may present neurological clinical signs (Gardiner et al., 1965; Nobre et al., 2004). Altered behaviours, such as dullness or hyperexcitability, head pressing against physical barriers, compulsive walking or circling and, occasionally, violent uncontrollable galloping are some of the findings in

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horses intoxicated by Crotalaria. Furthermore, intoxication can also cause decreased cranial nerve reflexes, ataxia and weakness (Nobre et al., 2004). Moreover, MCT and its metabolites have been found and quantified in the brain of rats experimentally intoxicated, which demonstrates the capacity of these molecules to cross the blood–brain barrier (Yan and Huxtable, 1995). Astrocytes are one type of glial cells and are essential to development, homeostasis and detoxification of the central nervous system (CNS) (Letournel-Boulland et al., 1994). They actively control synaptogenesis, synapse function, and synaptic plasticity (Barres, 2003), in addition to take up chemical transmitters released by neurons and maintaining extracellular ion levels (Aschner, 1998). Astrocytes are also part of the blood–brain barrier and may play a decisive role in biotransformation of xenobiotics (Tardy, 1991). Moreover, we previously demonstrated that these cells could react against chemical, traumatic, infectious, or toxic challenges (Costa et al., 2002; Pinheiro et al., 2006; Silva et al., 2007, 2008). The effects of MCT (and others PAs) upon different cell cultures have been clearly shown in previous studies (Hincks et al., 1991; Kim et al., 1995; Thomas et al., 1996; Pereira et al., 1998; Wilson et al., 1998; Kosogof et al., 2001). Furthermore, MCT metabolites can also alkylate cell macromolecules such as DNA (Wang et al., 2005; Xia et al., 2006) and the cytoskeletal protein actin (Wilson et al., 1998). In a pioneer study conducted by Barreto et al. (2006, 2008), it was observed that astrocytes react to MCT and DHMC by changing their morphology, disrupting and down regulating their major intermediary filament (IF), the glial fibrillary acidic protein (GFAP). These findings suggest a direct effect of MCT on this major glial cell type by a mechanism yet to be investigated. Although the activity of CYP450 mono-oxigenase system in the brain is only 1–10% of that of liver, small changes on its levels may have an important impact on brain function, including neurotransmission, development and behaviour, as well as in neurotoxicity (Warner and Gustafsson, 1995). Both microsomal and mitochondrial forms of CYP450 and its multiple family (including CYP450 3A) are normally expressed in brain of humans and in many animal species (Wilson et al., 2000; Ahlgren et al., 1990; Ravindranath et al., 1989, 1995, 2006; Strobel et al., 1995; Hagemeyer et al., 2003; Guengerich and Mason, 1979; Thomas et al., 1996; Reindel and Roth, 1991; Nebbia et al., 2003). Cytochrome P450 and its family have been reported in whole brain microsomal preparations (Bergh and Strobel, 1992) as well as in primary cultures of astrocytes and in glioma cell lines (Geng and Strobel, 1998; MalaplateArmand et al., 2005). Human glial cell lines have been established and characterised and constitute reliable and useful models to study either cell biology or diagnostic and pharmaceutical screenings (Pfeiffer and Betschart, 1978; Lal et al., 1996). The human GL-15 glioma cell line has been established and characterised by Bocchini et al. (1993). These cells express the astrocyte marker GFAP, and have been reported as a valid model to study the effects of xenobiotics on glial cells (Arcuri et al., 1995;ChambautGuerin et al., 2000; Planchenault et al., 2001; Costa et al., 2001, 2002; Hughes et al., 2005).

In this context, this study aimed to investigate the effect of MCT, extracted from C. retusa, on GL-15 cells, in terms of cytotoxicity, morphological changes, and induction of DNA damage and/or peroxidation of macromolecules. In addition, cytoskeletal proteins have been reported as potential targets for PAs (Wilson et al., 1998), therefore we also investigated the pattern and level of expression of the IF protein vimentin and the microtubule associated proteins (MAPs) on this cell model. 2. Material and methods 2.1. Cell line and culture GL-15 (Bocchini et al., 1993) stock cultures (passages 83–105) were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37  C in medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10% foetal bovine serum (GIBCO BRL, Grand Island, NY), a nutrient mixture (7 mM glucose, 2 mM glutamine, 0.011 g/l pyruvate), and in the presence of antibiotics (100 UI/ml penicillin G,100 mg/ml streptomycin). Cells were grown in 100 mm diameter tissue culture plates (TTP, Switzerland) containing 10 ml of medium, which was replaced three times per week. Stock cultures were passaged into new plates once every 3–4 days, and cultures for experiments were seeded into polystyrene culture dishes plates as needed. After 24 h the culture medium was changed and the different analytical procedures were performed on cultures. 2.2. Alkaloids extractions Monocrotaline was extracted and purified from an aqueous extract from C. retusa seeds according to Culvenor and Smith (1957) method as previously described (Barreto et al., 2006, 2008), except that hexane was used instead of petroleum ether and possibly N-oxide forms were not converted into free bases. The attainment of the DHMC was carried out by the technique described by Mattocks et al. (1989). Briefly, 20 mg of MCT was weighed and dissolved in 5 ml of chloroform. To this chloroform solution 5 ml of another chloroform solution was added containing 25 mg of 3,4,5,6-tetrachloro-1,2-benzoquinone (o-chloranil), the oxidant agent. After 2 min, this mixture was agitated vigorously for 15 s with 1 ml of a cooled solution of 70% KOH and 2% sodium borohydride, being the latter the reducing agent of the reaction. The organic phase was then transferred to a separation funnel, dehydrated with sodium sulphate (Na2SO4) and adsorbed with activated charcoal. The chloroform was removed at a rotavaporator, resulting in a practically pure dehydroalkaloid. The characterization of the MCT was carried out by 13C and 1H nuclear magnetic resonance, and infrared spectroscopy. The DHMC was confirmed by 1H nuclear magnetic resonance. 2.3. Treatments For treatments, MCT was dissolved in dimethylsulfoxide (DMSO, Sigma, St. Louis, MO) forming 100 mM stock solutions and stored at 20  C. DHMC was dissolved in

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dimethylformamide (DMF, Sigma, St. Louis, MO), immediately prior to experiments. Cells were treated with concentrations ranging between 0.1 and 500 mM MCT or DHMC for 24 and 72 h. The negative control group was treated with DMSO or DMF diluted in the culture medium at the higher equivalent volume used in treated groups (0.5%), and showed no significant effect on analysed parameters compared to cells that did not receive diluents. UV-C irradiation and hydrogen peroxide (0.3%) was adopted as positive control for genotoxicity and peroxidation tests, respectively. 2.4. Determination of macromolecular damages 2.4.1. Single cell electrophoresis assay of DNA (comet assay) DNA integrity and single-strand breaks were monitored using single cell gel electrophoresis (comet assay) performed under alkaline conditions essentially following the procedure described by Ribeiro et al. (2006) with some modifications. Briefly, all control and treated cells seeded on polystyrene culture dishes of 40 mm in diameter (1.5  105 cells/plate) were incubated for 24 or 72 h in the presence of 1–500 mM MCT or DHMC, 0.5% DMSO or 0.5% DMF (negative controls), or cells were exposed to UV-C irradiation for 1 h (positive control) at the wavelength of 253.7 nm, at a distance of 0.80 m from the source. After treatments the cells were scraped at 4  C and centrifuged at 1000g for 10 min. The pellets were diluted in 150 ml of PBS, and 30 ml of cell suspensions were mixed with 300 ml of 1% (w/v) low-melting-point agarose (Sigma–Aldrich), then they were applied on the surface of normal-meltingpoint agarose precoated-slides which were allowed to gel at 20  C for 5 min. Microgels were submersed in cell lysis buffer (14.61% NaCl, 3.72% EDTA, 0.12% Tris, pH 10.1%, Triton X-100, 10% DMSO) for 1 h at 4  C, protected from light. Following cell lysis, all slides were washed with PBS for 10 min to remove salt and detergent from the microgel. Slides were placed in a horizontal electrophoresis unit and were allowed to equilibrate for 20 min with electrophoresis buffer (0.034% EDTA, 0.3 N NaOH, pH 13). Electrophoresis was performed at 25 V for 20 min. Slides were rinsed three times with neutralisation solution (12.12% Tris–HCl, pH 7.5) for 5 min, fixed with 100% ethanol, air-dried, and stored protected from light until analysis. For analysis and scoring comets DNA in microgels were stained with 0.1% ethidium bromide (25 ml) for 5 min, and coverslips were applied before image analysis. Three replicate experiments were performed with two slides per experimental point. Cometassay samples were analysed at 200 magnification using an epifluorescent microscope (Olympus BX-2) and the Rhodamine filter. The image of the electrophoresed damaged DNA appears like a comet, with undamaged DNA in the head, and fragmented DNA migrating to form a tail. Comet images were recorded using a digital camera Variocam (PCO, Germany) connected to a personal computer. Two hundred randomly selected cells were scored from each slide (two slides per dose), and the percentage of comet cell (comet rate) was calculated. Tail length (comet length) of 100 randomly selected comet cells was also measured by a calibrated scale in the ocular of the microscope to evaluate the length of DNA migration (Speit et al.,

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1996; Silva et al., 2000; Boeira et al., 2001), using the software ImageJ 1.33u (Wayne Rasband, National Institute of Helth, USA). Comet tail length were scored based on an arbitrary scale of 0–4, which ranged from 0 (no damage) to 4 (with extensive damage of DNA). Thus, a group damage index could range from 0 (all nuclei without tail, 100 cells  0) to 400 (all nuclei with maximally long tails, 100 cells  4), according to Boeira et al. (2001). 2.4.2. Peroxidation of biomolecules Peroxidation of biomolecules induced by MCT was investigated by measuring thiobarbituric acid reactive substances (TBARS) (Draper and Hadley, 1990). GL-15 cells cultured in 100 mm diameter dishes (1 106 cells/plate) were incubated with MCT (1–500 mM) or 0.5% DMSO as a negative control. Peroxidation was induced in GL-15 cells by hydrogen peroxide (0.3%) and adopted as positive control. Four dishes were pooled and used for each experimental point, and three independent experiments were performed. Seventy-two hours after treatment the culture medium was removed and the cultures were rinsed three time with 5 ml phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 8.15 mM Na2HPO4, 1.47 mM KH2PO4, 0.492 mM MgCl2, 0.894 mM CaCl2, pH 7.4). The cells were trypsinized from the bottom of culture dishes, cell suspensions were centrifuged at 1359g for 10 min, at 4  C, and the pellet resuspended with 2.5 ml PBS. One millilitre of cell suspensions were transferred into glass centrifuge tubes, mixed with 2 ml of 0.67% (w/v) thiobarbituric acid in 15% (w/v) trichloroacetic acid and heated (100  C) for 15 min. After cooling to room temperature and centrifuged at 1359g for 5 min, the absorbance of the supernatant was measured at 535 nm in a spectrophotometer (Micronal B328). In parallel, the protein content per experimental point was measured in cell suspensions by the method of Lowry et al. (1951), using a protein assay reagent kit (Bio-Rad, Hercules, CA). The amount of TBARS was expressed as nmol malondialdehyde equivalents formed per mg cell protein. 2.5. Determination of morphological changes 2.5.1. Rosenfeld’s staining and immunocytochemistry Morphological changes were primarily assessed by analysing the Rosenfeld’s staining. All control and treated cells seeded on polystyrene culture dishes of 40 mm in diameter (1.5  105 cells/plate) were rinsed three times with PBS (without Ca2þ and Mg2þ) and fixed for 10 min with methanol at 20  C. Fixed cells were stained by the protocol established by Rosenfeld (1947). The Rosenfeld’s reagent (1 ml) was added and incubated for 20 min at room temperature. Thereafter, the plates were rinsed with water, air-dried, analysed in an optic phase microscope (Nikon TS-100) and photographed using a digital camera (Nikon E-4300). Morphological changes were also studied by phase contrast microscopy and by the immunocytochemistry patterns for the cytoskeletal proteins vimentin and MAPs (microtubule associated proteins). All control and treated cells seeded on polystyrene culture plates of 40 mm (1.5  105 cells/plate) were rinsed three times with PBS and fixed with cold methanol at 20  C for 10 min. Non-specific binding of antibody reagents was blocked by preincubating

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the plates with 3% bovine serum albumin (BSA) in PBS. Cells were incubated with anti-vimentin mouse monoclonal antibodies (clone 3B4, 1/250 in PBS, Biomakor, Israel) or rabbit polyclonal antibodies specific for MAPs (1/1000 in PBS, Sigma, St. Louis, MO) diluted in PBS, for 12 h at 4  C, under slow agitation. After three washes with PBS, the cells were incubated with rhodamine-conjugated antibodies specific for mouse or rabbit IgG (1/500 in PBS, Sigma, St. Louis, MO) diluted in PBS, for 2 h at room temperature. Thereafter, cells were analyzed using an epifluorescent microscope (Olympus BX-2) and images were recorded using a digital camera Variocam (PCO, Germany) connected to a personal computer. Always, 10 randomised representative fields were analysed. 2.5.2. Protein assay and westernblot Vimentin and MAPs expression were also investigated by western immunoblot. Total protein content was determined in control or treated cells seeded on polystyrene culture dishes of 40 mm in diameter (1.5  105 cells/plate). After 24–72 h exposure, cells were rinsed twice with PBS, lysed and harvested in a 2% (w/v) SDS, 2 mM ethylene glycol-bis[b-aminoethyl ether]-N,N,N0 N0 -tetraacetic acid (EGTA), 4 M urea, 0.5% (v/v) Triton X-100, 62.5 mM Tris–HCl buffer (pH 6.8), supplemented with 1 ml/ml of a cocktail of protease inhibitors (Sigma, St. Louis, MO). Protein content was determined by the method of Lowry et al. (1951), using a protein assay reagent kit (Bio-Rad, Hercules, CA). For western immunoblot analysis, 20 mg protein was loaded onto a discontinuous 4% stacking and 10% running SDS polyacrylamide gel (SDS-PAGE). Electrophoresis was performed at 200 V for 45 min. Proteins were then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA), at 100 V for 1 h. Equal protein loading was confirmed by staining the membranes with Ponceau Red (Sigma). Thereafter, membranes were blocked for 1 h at room temperature in 20 mM Tris-buffered saline (pH 7.5), containing 0.05% Tween 20 (TBS-T) and 5% powdered skim milk. Subsequently, membranes were incubated with mouse monoclonal antibodies anti-vimentin (1:1000, clone 3B4, OXFORD Biotecnology, Oxford, United Kingdom) or rabbit monoclonal antibodies anti-MAPs (1:1000, SIGNET, Dedham, MA) or for 1 h diluted in TBS-T containing 1% powdered skim milk. Conjugated alkaline phosphatase (AP) goat anti-rabbit IgG or goat anti-mouse IgG (1:5000 in TBS-T, Bio-Rad, Hercules, CA) were used as secondary antibodies. Immunoreactive bands were visualized using AP conjugated substrate Kit (Bio-Rad, Hercules, CA) according to manufacturer’s instructions. Quantification was obtained by scanning densitometry (ScanJet 4C – HP) of three independent experiments, and analyzed with ImageJ 1.33u (Wayne Rasband, National Institute of Health, USA). Antibody specificity and linearity of the densitometric analysis system was assessed by serial dilutions of total protein from cells in control conditions within a range of 5–20 mg of protein per lane. 2.6. Apoptosis detection Apoptosis was detected by monitoring the phosphatidylserine externalisation using an annexin V-Cy3/6-

carboxyfluorescein diacetate (6-CFDA) staining kit (Sigma– Aldrich, St. Louis, MO). 6-CFDA is used to measure viability; when this non-fluorescent compound enters living cells, esterases present hydrolyze it, producing the fluorescent compound 6-carboxyfluorescein (6-CF). Nontreated and treated cells were cultured for 24 h in 40 mm diameter plates (1.5  105 cells/plate). Cells were stained with 100 ml of binding buffer containing annexin V-Cy3 (1 mg/ml), 6-CFDA (500 mM), H2Odd and binding buffer (100 mM HEPES/NaOH, pH 7.5, 1.4 M NaCl, 25 mM CaCl2), following kit recommendations. After 15 min incubation at room temperature in the dark, cells were analysed using an epifluorescent microscope (Olympus BX-2) and images were recorded using a digital camera Variocam (PCO, Germany) connected to a personal computer. At least 10 fields were analysed and recorded, and the proportion of annexin-V/6-CF positive cells (which refers apoptosis) and annexin-V/6-CF negative cells (which refers necrosis) was determined. Apoptosis of GL-15 cells was also determined by the fluorescent dye Hoechst 33258 (Sigma–Aldrich, St. Louis, MO) staining, which allows to determine and quantify the cells with fragmented and condensed nuclear chromatin. Control and treated cells seeded on polystyrene culture plates of 40 mm (1.5  105 cells/plate) were rinsed three times with PBS and fixed with cold methanol at 20  C for 10 min. Chromatin was stained with Hoechst 33258 (5 mg/ml in PBS) for 10 min at room temperature in a dark chamber. Thereafter, cells were analysed using an epifluorescent microscope (Olympus BX-2) and images were recorded using a digital camera Variocam (PCO, Germany) connected to a personal computer. Each time, 10 randomised representative fields were analysed. The proportion of fragmented nuclei stained with Hoechst 33258 was determined in 10 microscopic fields for each experimental point. 2.7. Cytotoxic effects 2.7.1. Induction of membrane damages Membrane integrity was evaluated by measuring the lactate dehydrogenase (LDH) activity in culture medium of control and treated cells and evaluated as an index of cell damage. Cells were grown in 40 mm in diameter plates (1.5  105 cells/plate) treated with 1–100 mM MCT or with 0.5% DMSO (control), for 24–72 h. After that, the culture medium was removed and the LDH activity (UI/L) was measured according to manufacturer protocol (Doles, Goia´s, Brazil). Three independent experiments were carried out for each experimental point. 2.7.2. Cell viability The effect of MCT on the metabolism of GL-15 cells was tested using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, St. Louis, MO) test. The experiment was performed in 96-well plates (TPP Switzerland) (1 104 cells/well), and cells were incubated with 1–500 mM MCT or 0.5% DMSO (control) for 24 and 72 h. We also investigated a later MCT toxicity in GL-15 cells. In this case, after 72 h experiment the culture medium was changed (without MCT) and MTT test was performed after 6 days experiment. The cell viability was

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quantified by the conversion of yellow MTT by mitochondrial dehydrogenases of living cells to purple MTT formazan (Hansen et al., 1989). Control and treated cells were incubated with MTT at a final concentration of 1 mg/ml for 2 h. Thereafter, cells were lysed with 20% (w/v) sodium dodecyl sulphate (SDS), 50% (v/v) dimethylformamide (DMF) (pH 4.7), and plates were kept overnight at 37  C in order to dissolve formazan crystals. The optical density of each sample was measured at 492 nm using a spectrophotometer (Thermo Plate-Reader). Three independent experiments were carried out with eight replicate wells for each analysis. Results from MTT test were expressed as percentages of the viability of the treated groups related to the control groups. 2.8. Statistical analysis Results are expressed as mean  standard deviation. One-way ANOVA followed by the Student–Newmann– Keuls test was used to determine the statistical differences among groups differing in only one parameter. Student’s t-test was used to compare two groups. Values of P < 0.05 were considered as significant. 3. Results 3.1. Macromolecular damages GL-15 cells exposed to MCT were analysed for DNA strand breaks using Comet assay. The basal levels of DNA breaks (comet rate) observed in GL-15 cells cultures without any treatment were 4.5  0.45% (Fig. 1A). The frequency of DNA breaks observed in the cells in control conditions exposed to 0.5% DMSO was 5  0.52% and around 2.5  0.28% in cells exposed to 0.5% DMF. We observed, after 24 h treatment, that MCT induced a significant (P < 0.05) increase in the frequency of DNA breaks at any concentrations adopted (1–500 mM), which varied from 42  4.28% to 64  6.98%. On the other hand, no significant difference in the frequency of DNA breaks was observed in GL-15 cells exposed to 1–100 mM DHMC. However, exposure of the cells to 500 mM DHMC induced a significant increase (P < 0.05) in the frequency of DNA strand breaks, that reached 53  5.93%. As expected, the frequency of comets in the positive controls (UV irradiation) was 66  7.21%. The comet length was determined after measuring the comet tails in micrometers, and the index of DNA damage was determined as described in Section 2. As shown in Fig. 1B, after 24 h experiment, GL-15 cells without any treatment presented a low (9.84  1.2) basal index of DNA damage, as well as GL-15 cells in control conditions (0.5% DMSO and 0.5% DMF), indicating that the majority of cells presented no lesions on DNA (level 0). At these same conditions, only a small proportion of cells presented DNA strand breaks distributed at levels 1–3. In cells exposed to 1–500 mM MCT, we observed comets at all levels of lesions, whereas exposure to 500 mM DHMC caused most frequently DNA strand breaks of level 4. It reflected higher indexes of DNA damage caused by MCT when compared to control conditions, which ranged from 73.5  6.98 (1 mM MCT) to 176.8  19.2 (500 mM MCT) (Fig. 1B). Fig. 2 shows

Fig. 1. Determination of DNA strand breaks in GL-15 cells exposed to MCT and to the metabolite DHMC after 24 h treatment, using the comet assay. Control cells were exposed to the vehicles of alkaloid dilution (0.5% DMSO or 0.5% DMF), to the alkaloids (1–500 mM MCT or 1–500 mM DHMC), or to UV irradiation (positive control). (A) Comet rates (mean  SD) of at least 100 nuclei of GL-15 cells per experimental point, in three independent experiments; (B) indexes of DNA damage, based on comet tail length, calculated as described in Section 2. (*) Significant differences from respective control (0.5% DMSO or 0.5% DMF) values (P < 0.05).

examples of comets of different levels of DNA damage observed in GL-15 cells exposed to 500 mM MCT (C) or 500 mM DHMC (D), which were rarely evidenced in control conditions (A and B). The MCT property of inducing oxidative damage (peroxidation) of biomolecules was investigated after 72 h treatment, through the measurement of thiobarbituric acid reactive substances (TBARS). We observed that the production of TBARS in GL-15 cells exposed to MCT (1–500 mM) did not differ significantly from those observed in control conditions (0.5% DMSO), with TBARS values of 0.07  0.03 nmol/mg of protein (data not shown). This result indicates that MCT does not induce peroxidation of biomolecules. 3.2. Morphological changes and regulation of cytoskeletal components The morphology and immunostaining patterns of the cytoskeletal components vimentin and MAPs in GL-15 cells after 72 h treatment with (0.5%) DMSO (control) or with 10–500 mM MCT are shown in Fig. 4. In control conditions, as revealed by phase microscopy, Rosenfeld’s staining and

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Fig. 2. Images of comets observed after 24 h treatment of GL-15 cells exposed to the vehicle of alkaloid dilution 0.5% DMSO (A) and 0.5% DMF (B), or exposed to the alkaloids 500 mM MCT (C) and 500 mM DHMC (D). The DNA strand breaks were observed by fluorescent microscopy after ethidium bromide staining. Obj. 20  0.70; scale bars ¼ 10 mm.

also by vimentin immunostaining (Fig. 3A, E and I, respectively), GL-15 cells have a bipolar fibroblast like phenotype, whose size varies to some extent depending on the density of the culture. A single exposure of cultures of GL-15 cells to 1–10 mM MCT produced megalocytosis beginning by 24 h (data not shown). This phenotype, characterised by markedly enlarged cells and nuclei in glial cells exposed to 10 mM MCT, was clearly evident after 3 days (Fig. 3B, F, and J). Moreover, megalocytotic cultures remained confluent, presenting a normal adhesion of cells and a radial distribution on vimentin pattern (Fig. 3J). On the other hand, GL-15 cells treated with 100 mM MCT became much more elongated, with some cells featuring a retracted cytoplasm and thin cellular processes (Fig. 3C, G and K). This effect was more pronounced after treatment with the highest concentrations of MCT (500 mM) (Fig. 3D, H, and L). Vimentin immunolabelling in GL-15 cells, performed after 72 h experiment, showed that this IF protein was condensed and restricted to the cell body after exposure to 100 mM MCT (Fig. 3K). In addition, aggregates of vimentin were evidenced in cells exposed to 500 mM MCT (Fig. 3L). Changes in the immunocytochemistry pattern of MAPs were also investigated in GL-15 cells after exposure to MCT (Fig. 3M–P). In cells under control conditions (0.5% DMSO), MAPs appear as punctual immunolabelling showing some aggregates (Fig. 3M). A similar pattern of immunolabelling was also evident after 72 h experiment in GL-15 cells exposed to the highest MCT concentrations adopted

(100–500 mM, Fig. 3O and P, respectively). However, MAPs immunolabelling was low in GL-15 cells exposed to 10 mM MCT (Fig. 3N), and it was almost absent in GL-15 cells exposed to 1 mM MCT (data not shown). The effect of MCT, regarding expression of vimentin and MAPs by cells, was also assessed by western blotting after 72 h experiment (Fig. 4). Immunodetection of MAPs in protein extracts from GL-15 cells in control conditions (0.5% DMSO) revealed a more intense immunoreactive band of 68 kDa and another immunoreactive band, of lowest intensity, of 57 kDa. However, it was observed that GL-15 exposure to MCT diminished MAPs expression, especially in protein extracts from cells exposed to 1–10 mM MCT (Fig. 4A). The intermediate filament vimentin is constitutively expressed in GL-15 cells at relatively high levels, and an immunoreactive band of 55 kDa could be clearly detected by western immunoblot of protein extracts obtained from cells in control conditions (0.5% DMSO) (Fig. 4B). An immunoreactive band of similar intensity was observed in protein extracts obtained from GL-15 cells treated with 1 mM MCT. However, vimentin protein was slightly decreased in protein extracts obtained from cells exposed to 10–500 mM MCT. 3.3. Apoptosis We investigated by annexin V-Cy3/6-CFDA the capability of MCT to induce apoptosis or necrosis on glioblastoma cells (Fig. 5). After 24 h experiment in control

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Fig. 3. Photomicrographs of GL-15 cell cultures in control conditions (0.5% DMSO) or after 72 h exposure to 10–500 mM MCT. (A–D) Photomicrographs by phase microscopy; obj. 20  0.70; (E–H) photomicrographs after staining by Rosenfeld’s staining; obj. 20  0.70; (I–L) photomicrographs after immunocytochemistry for the intermediary filament protein vimentin; obj. 20  0.70; (M–P) photomicrographs after immunocytochemistry for the microtubule associated protein (MAPs); obj. 100  0.70. Scale bars ¼ 10 mm.

conditions (0.5% DMSO), the proportion of annexin V-Cy3/ 6-CF labelled cells (cell in apoptosis) was less than 1% and annexin V-Cy3 appeared as very small spots. In contrast, the proportion of annexin V-Cy3/6-CF positive cells exposed to 500 mM MCT was around 50%, indicating induction of apoptosis. MCT at this concentration did not modify the percentage of necrotic GL-15 cells (annexin V-Cy3 positive/6-CF negative cells) when compared to control. Moreover, nuclear condensation and fragmentation of DNA, features that characterise apoptosis, were observed by Hoechst 33258 nuclear staining of adherent cells. As shown in Fig. 5D, apoptosis signs were observed after a 72 h exposure to 500 mM MCT, in 40% of total remaining cells.

3.4. Cytotoxicity The effects of MCT, extracted from C. retusa, upon the cell viability were assessed by the measurement of lactate dehydrogenase (LDH) on the cell culture medium and by measuring the mitochondrial function using the MTT test. Although some variations were observed in cultures exposed to MCT (1–500 mM) after 24 or 72 h, both LDH and MTT assays showed no significant cytotoxic effects towards GL-15 cells. However, when the MTT test was performed after a longer time of exposure (6 days), a significant (P < 0.05) cytotoxic effect was evidenced in GL-15 cells exposed to 500 mM MCT (data not shown).

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Fig. 4. Modulation of the microtubule associated proteins (MAPs) and intermediary filament vimentin expression by the alkaloid MCT in GL-15 cultures. Immunoreactive bands after western blot analysis of MAPs (68/ 57 kDa) (A) and vimentin (55 kDa) (B) proteins’ expression in GL-15 cells in control conditions (0.5% DMSO) or after 72 h exposure to 1–500 mM MCT. Samples containing 20 mg of total protein were electrophoretically separated in 10% polyacrylamide gels containing 0.1% SDS in running buffer. Histogram represents the relative expression of MAPs and vimentin proteins as arbitrary densitometric units. Data are representative of three individual experiments.

4. Discussion MCT metabolites have been found and quantified in the brain of experimentally intoxicated rats, which demonstrates the capacity of these molecules to cross the blood– brain barrier (Yan and Huxtable, 1995). Moreover, more susceptible animals to this intoxication, such as horses, may present neurological signs after intoxication with Crotalaria (Nobre et al., 2004). Glial cells, and mainly astrocytes, the major cells of the glial family, are essential for the nutritional and structural support to neurons in the CNS. They also participate in other functions such as immune response and cerebral detoxification, featuring an

active CYP450 system (Tardy, 1991; Letournel-Boulland et al., 1994; Montgomery, 1994). Thus, modifications on astrocytes may constitute biological markers for many types of damages in the CNS. In our previous studies in an in vitro model of rat astrocyte primary cultures, we observed that that DHMC was cytotoxic to astrocytes at low concentrations such as 1 mM after 24 h (Barreto et al., 2008), meanwhile MCT was not cytotoxic even after a 72 h exposure to 500 mM (Barreto et al., 2006). MCT and DHMCT also altered the morphology of the cells with changes on the expression pattern of GFAP, the main component of IF in astrocytes, suggesting a potential involvement of these cells on neurological damages observed in intoxicated animals. In this context, we adopted the human cell line GL15 as an in vitro model of glial cells, in order to bring new knowledge about the mechanisms of cytotoxicity of this important vegetal toxin in CNS cells, as well as its relation with the pathogenesis of PAs intoxication. We observed by the MTT test and by measuring the LDH activity on the culture medium of cells that the alkaloid MCT did not induce changes on cell viability even after 72 h exposure at the concentrations adopted (1–500 mM). However, the genotoxic property of MCT, evidenced by the Comet test, showed that exposure of cells to 1–500 mM MCT caused a significant and dose-dependent increase in DNA damage, which reached 42–64% as soon as after 24 h exposure. Interestingly, we observed that GL-15 cells exposed to the metabolite DHMC, at concentrations ranging between 1 and 100 mM, did not present the same significant levels of DNA damage observed after MCT treatment. On the other hand, a significant DHMC genotoxic effect was observed when this drug was adopted at a higher concentration (500 mM). It has been proposed that DHMC is a highly unstable intermediate reactive metabolite of MCT, which can (i) undergo hydrolysis to form ()6,7-dihydro-7hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) followed by reaction with DNA to form DHP-adducts, (ii) bind to cellular DNA followed with hydrolysis to form the DHPderived DNA adducts, or (iii) conjugate with cellular proteins and glutathione (Petry et al., 1984; Yan and Huxtable, 1995). Although the half-life of DHMC has been established at 3.4 s (Cooper and Huxtable, 1996), this compound has been administered in vivo to induce pulmonary hypertension 8 weeks after the injection (Okada et al., 1995). Furthermore, we previously showed that astrocytes are sensible to DHMC at low concentrations (1 mM) (Barreto et al., 2008). Although the main generation of DHP results from the formation of DHMC catalysed by P450 followed by hydrolysis, other pathways, such as the DHP formation from the dehydrogenation of retronecine, the hydrolysis product of MCT, cannot be excluded (Lin et al., 2002; Wang et al., 2005). Another fate of MCT and others PAs is to be metabolised by P450, forming the derivative MCT N-oxide (Mattocks and White, 1971; Mattocks and Bird, 1983; Taylor et al., 1997). PA N-oxides are highly water-soluble and have been generally regarded as detoxification products (Mattocks, 1971a,b, 1986; Mattocks and White, 1971; Jago et al., 1970; Phillipson, 1971). Some PA N-oxides, such as indicine N-oxide have been shown to have anti-mitotic and anti-tumour activities (Kugelman et al., 1976). Others PA N-oxides, such as MCT, lasiocarpine, and

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Fig. 5. Determination of apoptosis induced by MCT in GL-15 cells. Annexin V-Cy3/6-CFDA staining performed after 24 h experiment to detect apoptosis or necrosis induced by MCT in GL-15 cells in control conditions (0.5% DMSO) (A) or treated with 500 mM MCT (B), indicating annexin V-Cy3/6-CF labelled apoptotic cells. Hoechst 33258 staining of nuclear chromatin in cultures 72 h post-treatment in control condition (0.5% DMSO) (C), and in cultures of cells treated with 500 mM MCT (D) showing nuclear condensation and fragmentation. Obj. 200.70, scale bars ¼ 10 mm.

fulvine N-oxides, have been reported to be as toxic in laboratory animals as the corresponding PAs (Mattocks, 1971b). In addition, Chou et al. (2003) found DHP-derived DNA adducts in calf thymus DNA incubated with liver microsomal system and riddelliine N-oxide, as well as in liver DNA of F344 rats fed with riddelliine N-oxide or riddelliine. These authors also found that levels of DHP-DNA adducts were correlated with the index of liver tumour formation, indicating that riddelliine N-oxide is also a potential genotoxic hepatocarcinogen. In this study, cells exposed to DHMC may not have the capacity to generate MCT N-oxide, and we may presume that it was a limiting factor in metabolism of DHMC to DHP in order to form DHPderived DNA adducts. A small proportion of cells with DNA damage were observed in GL-15 cells in control conditions, although, as described by Tice et al. (2000), it may be due to the high sensitivity of the comet test, which may detect DNA damages at very low levels. Similar frequencies of DNA damage in control conditions have also been described in other in vitro studies with genotoxic alkaloids (Boeira et al., 2001), and probably it is due to the characteristics of the study’s systems that uses tumour cell lines. It is well established that the pyrrolic metabolite (DHP) is conjugated with glutathione (GSH), forming a glutathioneconjugated pyrrolic metabolite of 6,7-dihydro-7-hydroxy1-hydroxymethyl-5H-toxin pyrrolizine (GSH-DHP), which is water-soluble and more stable than DHP in aqueous environments (Lame´ et al., 1990). Generally, glutathione conjugation is considered to be a detoxification process by which xenobiotics and other compounds are eliminated

(Baillie and Kassahun, 1994). The presence of relatively lows levels of the endogenous nucleophile glutathione in brainstem (Ravindranath et al., 1989) may render cells particularly vulnerable to damage through CYP450-mediated bioactivation of xenobiotics to reactive electrophilic metabolites, such as DHP. In this view, in our system, the dose-dependant increase in genotoxicity induced by MCT may be due to depletion of the endogenous nucleophile glutathione in GL15 cells, rendering these cells more vulnerable to large amounts of MCT pyrrole (MCTP) that probably may not be conjugated with glutathione. To bear this hypothesis, a study blocking endogenous nucleophile glutathione in GL-15 cells with antagonists before exposure to low, but genotoxic levels of MCT must be developed. The annexin-Cy3/6-CFDA staining and the nuclear chromatin stain by Hoechst 33258 dye revealed both phosphatidylserine externalisation and condensed fragmented chromatin in GL-15 cells treated with 500 mM MCT. As these are events that characterise apoptosis (Heatwole, 1999) we may suppose that MCT induces apoptosis in GL15 cells. Apoptosis has also been found in hepatic and pulmonary vascular endothelial cells exposed to MCT or its DHP in vivo (Jones and Rabinovitch, 1996) and in vitro (Thomas et al., 1996, 1998; Copple et al., 2004). In this study we demonstrated that GL-15 cells also reacts to MCT exposure undergoing phenotypical changes depending on the concentration adopted. Exposure of GL-15 cells to low concentrations of MCT (1–10 mM) clearly induced hypertrophy and megalocytosis, as evidenced by phase microscopy, Rosenfeld staining and also by vimentin

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immunocytochemistry. Hypertrophy caused by MCT has also been described in in vivo and in vitro primary cellular targets, such as, hepatocytes, vascular smooth muscle, and breast and pulmonary artery endothelial cells (TodorovichHunter et al., 1988; Wilson and Segall, 1990; Thomas et al., 1996; Taylor et al., 1997; Wilson et al., 1998, 2000; Mathew et al., 2004; Mukhopadhyay et al., 2006). According Mukhopadhyay et al. (2006), this phenotype is characterised by development of markedly enlarged cells with enlarged nuclei and ‘‘proliferation’’ of intracellular organelles including enlarged Golgi, increased endoplasmic reticulum and vacuolation. In fact, we observed that hypertrophic GL-15 cell presented enlarged and multiple nuclei and also some vacuolation could be observed. Enlarged astrocytes with multiple nuclei were also observed in our previous study when cells were exposed to 1–10 mM MCT (Barreto et al., 2006). On the other hand, GL-15 cells exposed to 100 mM MCT presented a contracted cytoplasm with thin cellular processes and vimentin labelling limited to the cell body. This effect was apparently amplified when cells were exposed to 500 mM MCT and vimentin labelling appeared ‘‘as packets’’ with a more perinuclear location. This phenomenon was associated with changes in vimentin protein steady state level, as revealed by western immunoblot. At the intermediary concentrations (10–100 mM), only a slight but not significant decrease in vimentin levels was observed, which suggests a vimentin disturbance more than a down regulation itself. Vimentin is the major IF of the cytoskeleton of undifferentiated glial cells such as the GL-15 glioma cells (Stichel et al., 1991; Bocchini et al., 1993; Bohn et al., 1993; Costa et al., 2001), and its assembly as polymers has an important role in determining the cellular shape. Considering that PAs are able to alkylate macromolecules like the cytoskeletal protein actin (Wilson et al., 1998), we infer that MCT may be interacting with vimentin, changing its assembly and stability. Taken together, our results indicate that the IF from glial cells are potential targets for MCT, which is in accordance with the findings of a previous study on astroglial cells in primary cultures (Barreto et al., 2006, 2008) showing that GFAP expression in astrocytes are also down regulated after treatment with MCT or DHMC. The effect of MCT on microtubule-associated proteins (MAPs), another important cytoskeletal component of CNS cells, was also investigated. MAPs have been identified in many different cell types and they have been found to carry out a wide range of functions. These functions include both stabilising and destabilising microtubules, guiding microtubules towards specific cellular locations, cross-linking microtubules and mediating the interactions of microtubules with other proteins in the cell (Alberts et al., 2002). Classical MAPs, such as MAP2 and Tau, have been shown to stabilise microtubules by binding to the outer surface of the microtubule protofilaments (Heald and Nogales, 2002). In an adult, MAP2 expression is rarely found in non-neuronal cells, but has been described in astrocytes from the optic nerve and infundibulum (Papasozomenos and Binder, 1986), in areas of gliosis (Saito et al., 1988), in Bergmann’s fibbers from the cerebellum of birds (Tucker et al., 1996), and in the C6 rat glioma cell line (Garner et al., 1988; Garner and Matus, 1988). More recently, it was demonstrated that

multi-potent precursory cells of the nervous system of rats co-express MAP-2 and GFAP (Rosser et al., 1997), and its role in the development has been widely studied and demonstrated. These findings demonstrate that mature astrocytes and undifferentiated glial cells keep the capacity to express MAP-2 under certain conditions. In this study, we observed through western blot and immunocytochemistry that GL-15 cells express MAPs. Using polyclonal antibodies, that recognise both MAP-2 and Tau, we verified that these proteins are distributed in the cellular body as small spots, and, sometimes, as aggregates. Moreover, two immunoreactive bands of 57 and 68 kDa were detected by western blot in protein extracts of these cells. Interestingly, we observed an important down-regulation in the expression of MAPs in cells exposed to 1–10 mM MCT. However, this negative regulation of the MAPs was less evident in the cells exposed to the higher MCT concentrations (100–500 mM). The low levels of MAPs expression were associated with the hypertrophy and multiplicity of nuclei in GL-15 cells exposed to the lowest concentrations of MCT (1–10 mM). These findings suggest that MCT interferes with the dynamics of the growth and cellular proliferation, through regulation of factors of stabilisation of microtubules like the MAPs, possibly inducing intracellular mechanisms of signalling. These effects were not evident in GL-15 cells exposed to the higher MCT concentrations (100–500 mM). In case of a strong genotoxicity, we might suggest that, despite the fact that regulatory mechanisms may be activated inducing a down-regulation of the MAPs, they are not efficiently concluded. Therefore, very important injuries in the DNA of the cells can induce others routes of signalling leading to different outcomes, such as apoptosis, which was evidenced in our investigation. Toxicity induced by PAs leads to oxidative damages and has also been related to a longer known recruitment of inflammatory cells during the MCT pneumotoxicity (Stenmark et al., 1985). On the other hand, it has been demonstrated that antioxidants can attenuate oxidative damages during MCT-induced cytotoxicity (Chen et al., 2001). It has been shown that phase I drug metabolism in the brain is manly done by cytochrome P450 (Ravindranath et al., 2006), resulting in the formation of hydrophilic metabolites that are removed by renal clearance. On the other hand, derived metabolites from drugs, such as phenytoin derivatives and the alkaloid tetrahydropapaveroline, can exert their cellular toxicity by covalent binding to cellular components, causing membrane disorders and resulting in oxidative damage to proteins and lipids (Vorhees et al., 1990; Liu and Wells, 1994; Meyer et al., 2001; Soh et al., 2003). We observed according to the MTT and LDH tests that MCT was not toxic to GL-15 cells after 24–72 h exposure, but it changed cell morphology, the organisation and expression of cytoskeletal components, such as vimentin and MAPs. These results corroborate our previous findings in primary astrocyte cultures (Barreto et al., 2006, 2008). However, toxicity was observed later in older cultures (6 days), indicating that cumulative genotoxic damages disturb viability of GL-15 cells. It was also observed in this study that at our experimental conditions MCT metabolism by GL-15 glial cells did not induce peroxidation of biomolecules, suggesting again that its toxic effect may be

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mainly due to destabilisation of structural proteins and genotoxicity. Furthermore, as yet discussed, it is well known that this is not MCT, but rather its metabolites such as DHMC, MCT pyrrole and MCT-N-oxides that are responsible for the cytotoxic effects in different cell systems. In this view, the sensibility of GL-15 cells to this alkaloid suggests that this human cell linage has an efficient system of the activation of xenobiotics to toxic metabolites, and may constitute a good in vitro model to detect metabolismmediated toxicity in glial cells. Acknowledgements This work was supported in part by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and Fundaça˜o de Amparo a` Pesquisa e Extensa˜o do Estado da Bahia (FAPESB). We gratefully acknowledge the research support provided by Programa de Pos-graduaça˜o em Cieˆncia Animal dos Tro´picos/UFBA, and Fundaça˜o Coordenaça˜o de Aperfeiçoamento de Pessoal de Nı´vel Superior (CAPES). Conflict of interest The authors declare that there are no conflicts of interest. References Ahlgren, R., Warner, M., Gustaffson, J.A., 1990. Cloning of a rat P45026hydroxylase cDNA, comparison of tissue distribution with rat P450scc. In: Ingelman-Sundberg, M., Gustaffson, J.A., Orrenius, S. (Eds.), Drug Metabolizing Enzymes: Genetics, Regulation and Toxicology. Karolinska Institute, Stockholm, pp. 158–161. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P., 2002. Molecular Biology of the Cell, fourth ed. Garland Science Publishing, New York. 909–982. Alfonso, H.A., Sanchez, L.M., Figueredo, M.A., Go´mez, B.C., 1993. Intoxication due Crotalaria retusa and C. spectabillis in chickens and geese. Veterinary Human Toxicology 32, 539. Arcuri, C., Bocchini, V., Guerrieri, P., Fages, C., Tardy, M., 1995. PKA and PKC activation induces opposite glial fibrillary acidic protein (GFAP) expression and morphology changes in a glioblastoma multiform cell line of clonal origin. Journal of Neuroscience Research 40, 622–631. Aschner, M., 1998. Astrocytes as mediators of immune and inflammatory responses in the CNS. Neurotoxicology 19, 269–282. Baillie, T.A., Kassahun, K., 1994. Reversibility in glutathione-conjugate formation. Advances in Pharmacology 27, 163–181. Barres, B.A., 2003. What is a glial cell? Glia 43, 4–5. Barreto, R.A., Hughes, J.B., Souza, C.S., Silva, V.D.A., Silva, A.R., Velozo, E.S., Batatinha, M.J.M., Costa, M.F.D., El-Bacha´, R.S., Costa, S.L., 2006. The pyrrolizidine alkaloid monocrotaline, extracted from Crotalaria retusa, interferes on cellular growth, alters GFAP expression and induces morphological changes on astrocyte primary cultures. Brazilian Journal of Health and Animal Production 7, 112–127. Barreto, R.A., Sousa, C.S., Silva, V.D.A., Silva, A.R., Veloso, E.S., Cunha, S.D., Costa, M.F.D., El-Bacha´, R.S., Costa, S.L., 2008. Monocrotaline pyrrol is cytotoxic and alters the patterns of GFAP expression on astrocyte primary cultures. Toxicology in Vitro 22, 1191–1197. Bergh, A.F., Strobel, H.W., 1992. Reconstitution of the brain mixed function oxidase system: purification of NADPH-cytochrome P450 reductase and partial purification of cytochrome P450 from whole rat brain. Journal of Neurochemistry 59, 575–581. Bocchini, V., Beccari, T., Arcuri, C., Bruyere, L., Fages, C., Tardy, M., 1993. Glial fibrillary acidic protein and its encoding mRNA exhibit mosaic expression in a glioblastoma multiform cell line of clonal origin. International Journal of Developmental Neuroscience 11, 485–492. Boeira, J.M., da Silva, J., Erdtmann, B., Henriques, J.A., 2001. Genotoxic effects of the alkaloids harman and harmine assessed by comet assay

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