Monocrotaline: Histological Damage and Oxidant Activity in Brain Areas of Mice

This work was designed to study MCT effect in histopathological analysis of hippocampus (HC) and parahippocampal cortex (PHC) and in oxidative stress (OS) parameters in brain areas such as hippocampus (HC), prefrontal cortex (PFC), and striatum (ST). Swiss mice (25–30 g) were administered a single i.p. dose of MCT (5, 50, or 100 mg/kg) or 4% Tween 80 in saline (control group). After 30 minutes, the animals were sacrificed by decapitation and the brain areas (HC, PHC, PFC, or ST) were removed for histopathological analysis or dissected and homogenized for measurement of OS parameters (lipid peroxidation, nitrite, and catalase) by spectrophotometry. Histological evaluation of brain structures of rats treated with MCT (50 and 100 mg/kg) revealed lesions in the hippocampus and parahippocampal cortex compared to control. Lipid peroxidation was evident in all brain areas after administration of MCT. Nitrite/nitrate content decreased in all doses administered in HC, PFC, and ST. Catalase activity was increased in the MCT group only in HC. In conclusion, monocrotaline caused cell lesions in the hippocampus and parahippocampal cortex regions and produced oxidative stress in the HC, PFC, and ST in mice. These findings may contribute to the neurological effects associated with this compound.


Introduction
Monocrotaline (MCT) is a pyrrolizidine alkaloid produced by Crotalaria genus, which causes hepatotoxic effects in animals and man [1,2]. Pyrrolizidine alkaloids are activated in vivo by liver cytochrome P450 mixed-function oxidases, producing generation of electrophilic pyrrolic intermediates [3]. Dehydromonocrotaline appears to be detoxified by conjugation with glutathione (GSH) [4]. Dehydromonocrotaline inhibits the activity of the respiratory chain complex I NADH oxidase. The probable mechanism is a change in the conformation of complex I resulting from modification of cysteine thiol groups by the metabolite [5]. Humans are exposed to MCT following consumption of herbal teas or contaminated food grains [1,6]. Lethal toxicity has been reported in sheep 38-120 h after administration of Crotalaria retusa 5-40 g/kg [7,8]. Horses are particularly sensitive to intoxication by MCT and show classic picture of liver fibrosis and neurological symptoms (Kimberley horse disease) with chronic exposure to the alkaloid [9,10]. Preliminary studies show that MCT causes lesions in some organs, such as liver [11] and lungs [12]. There are also reports of histological changes in these organs and MCT neurotoxicity is associated with its lipophilicity [9,13], but there is a little information about the associated histological changes in the central nervous system (CNS).
Oxidative stress (OS) is classically defined as a redox imbalance caused by an excess of oxidants or a lack of natural antioxidants in the system [14,15]. The normal brain consumes higher rate of oxygen per unit mass of tissue and contains low levels of antioxidants. There are excitatory amino acids in normal brain, and these amino acids are important to normal function. Also, neurotransmitters are molecules involved with oxidation process. For example, dopamine and noradrenaline react with molecular oxygen to form quinines and semiquinone, which can deplete glutathione [16,17]. Likewise, in brain, the oxidation of dopamine by monoamine oxidase releases hydrogen peroxide as a metabolic product that can cause tissue injury, including lipid peroxidation, DNA damage, and enzyme inactivation [18]. Brain lipids are highly enriched with polyunsaturated fatty acids [16,19]. In addition, certain regions of the central nervous system, such as striatum and hippocampus, may be particularly sensitive to OS because of their low endogenous level of vitamin E, an important biochemical antioxidant, relative to other brain regions [20]. Some studies with MCT observed that the levels of glutathione (GSH) are more than 50% increase in rat liver after MCT (65 mg/kg, i.p.) administration [4]. On the other hand, MCT increases the intensity of cellular OS in pulmonary artery endothelial cells [1,12]. Based on the facts cited above and that MCT administration to mice caused CNS alterations [21], we decided to assess the effect of MCT administration in histopathological analysis as well as in tests of oxidative stress through thiobarbituric acid reactive substances (TBARS), as an indicator of lipid peroxidation, as well as catalase activity and nitrite/nitrate content in the hippocampus, prefrontal cortex, and striatum. The idea is to study the relation of ROS and reactive nitrogen species (RNS) with MCT neurotoxic effects.

Extraction and Isolation of Monocrotaline.
The ground seeds (640 g) of C. retusa were extracted three times with 95% ethanol for 72 h. The ethanolic extracts were evaporated under reduced pressure to yield a brown viscous residue (53.4 g), which was extracted with 3% hydrochloric acid until the washing was negative to Dragendorff 's reagent. The acidic aqueous solution was made alkaline (pH 9) with ammonia (25%) and repeatedly extracted with chloroform. The chloroformic solution was dried with Na 2 SO 4 anhydrous, filtered, and evaporated until dryness to obtain the total alkaloid fraction (10.48 g). Analytical thin layer chromatography showed the presence of a single alkaloid. The dried residue was recrystallized from ethanol to give colorless prismatic crystals (yield ca. 1.4% based on the dry weight of the seeds material), which melted at 197-198 • C. It was identified as monocrotaline based on 1H NMR, 13C NMR, HMQC, and HMBC spectroscopic data and comparison with those reported by Barreiro et al. (1980) and Cheng et al. (1986) [22,23].
Animals were treated once with MTC at the doses of 5, 50, or 100 mg/kg i.p. and divided into 3 groups of 10 animals each. Control groups (vehicle) received 4% Tween 80 dissolved in saline 0.9% at the same volume as treated animals (10 mL/kg). Twenty-four hours after the drug administration the animals were sacrificed and their brain areas (hippocampus-HC, parahippocampal cortex-PHC, prefrontal cortex-PFC, or striatum-ST) were removed for histological analysis or dissected and homogenized with 10% phosphate buffer 0.05 M pH 7.4 for OS (HC, PFC, or ST) parameters determinations.

Histopathological
Analysis. The brains were rapidly dissected, fixed in 4% neutral-buffered paraformaldehyde, and processed routinely for paraffin embedding. Sagittal sections, 5 µm thick, were cut serially at the level of the hippocampus, parahippocampal cortex, and selected sections were stained with hematoxylin and eosin (H/E) for light microscopy examination. The histological observations (evaluated by a pathologist using a double-blind method) were scored using a pathological scoring scale modified in work of Gilat  We included immunohistochemistry analysis in our results. Immunohistochemistry for caspase-3 was performed using the streptavidin-biotin-peroxidase method [26] in formalin-fixed, paraffin-embedded tissue sections (4 µm thick), mounted on poly-L-lysine-coated microscope slides. The sections were deparaffinized and rehydrated through xylene and graded alcohols. After antigen retrieval, endogenous peroxidase was blocked (15 min) with 3% (v/v) hydrogen peroxide and washed in phosphate-buffered saline (PBS). Sections were incubated overnight (4 • C) with primary anti-caspase-3 antibody (polyclonal goat anti-mouse) diluted 1 : 200 in PBS plus bovine serum albumin (PBS-BSA). The slides were then incubated with biotinylated goat anti-IgG, diluted 1 : 200 in PBS-BSA. After washing, the slides were incubated with avidin-biotin-horseradish peroxidase conjugate (Strep ABC complex by Vectastain ABC reagent and peroxidase substrate solution) for 30 min, according to the Vectastain protocol. Caspase-3 was visualized with the chromogen 3,3 diaminobenzidine (DAB). Negative control sections were processed simultaneously as described above but with the first antibody being replaced by PBS-BSA 5%. None of the negative controls showed caspase-3 immunoreactivity. Slides were counterstained with Harry's hematoxylin, dehydrated in a graded alcohol series, cleared in xylene, and coverslipped.

Measurement of Lipid Peroxidation.
Lipid peroxides formation was analyzed by measuring the thiobarbituricacid reacting substances (TBARS) in the homogenates [27]. The samples were added to a catalytic system of formation of free radicals (FeSO 4 0.01 mM and ascorbic acid 0.1 mM) and then incubated at 37 • C for 30 min. The reaction was stopped with 0.5 mL of 10% trichloroacetic acid, then the samples were centrifuged (3000 rpm/15 min), and the supernatants were retrieved and mixed with 0.5 mL of 0.8% thiobarbituric acid then heated in a boiling water bath for 15 min and after this period, they were immediately kept cold in a bath of ice. Lipid peroxidation was determined by the absorbance at 532 nm and was expressed as µmol of malondialdehyde (MDA)/g tissue.  [27]. The activity was measured by the degree of this reaction. The assay mixture contained 0.3 mL of hydrogen peroxide in 50 mL of 0.05 M phosphate buffer, pH 7.0. The sample aliquot (20 µL) was added to 980 µL of the substrate mixture. Initial and final absorbances were recorded at 230 nm after 1 and 6 min, respectively. A standard curve was established using purified catalase (Sigma, MO, USA) under identical conditions. All samples were diluted with 0.1 mmol/L phosphate buffer (pH 7.0) to provoke a 50% inhibition of the diluent rate (i.e., the uninhibited reaction). Protein was determined using bovine serum albumin as standard [29]. Results were expressed as mM/min/mg protein.

Discussion
The alkaloids are molecules that may cause hepatotoxic, pneumotoxic, mutagenic, and neurotoxic effects [4]. Kimberley horse disease is observed in horses intoxicated with monocrotaline (pyrrolizidine alkaloid) and it presents neurological symptoms which have been associated with neural alteration [30]. On this way, Nobre et al. observed that animals intoxicated with MCT showed clinical symptoms related to CNS such as depression, decreased locomotor activity, and motor incoordination [9,24].
Our histopathological results showed that 50 or 100 mg/kg of monocrotaline caused lesions in the hippocampus and parahippocampal cortex regions. Lesions observed in our work were severe structural disorganization, edema, moderate pyknotic cells, vacuolization, and inflammatory cell infiltration. These results are according to previous studies that showed vacuolization and increase in cell body in cortical astrocyte/neuron primary coultures, referring to neuronal damage induced by MCT [31]. Similarly, another study showed that pyrrolizidine alkaloid monocrotaline induced cytotoxicity, morphological changes, and oxidative and genotoxic damages to glial cells, using the human glioblastoma cell line GL-15 as a model [32]. The results of monocrotaline immunochemistry in two doses showed an increase of caspase 3 in brain areas compared to control, indicating apoptosis, so these results are according to histological data. Apoptosis is characterized by DNA fragmentation, membrane blebbing, cell shrinkage, and disassembly into membrane-enclosed vesicles, which prevents a host inflammatory response to intracellular components [33]. At molecular level, classical apoptosis is caused by the activation of a family of cysteine protease known as caspases that cleave their target protein at specific aspartic acids. Two major apoptotic pathways culminate in the activation of downstream executioner caspases 3 and 6 [34,35]. So caspase 3 is an immunohistochemistry marker to apoptosis.
Based on these histological alterations in hippocampus and parahippocampal cortex regions and behavioral studies that demonstrated that MCT is able to cause serious alterations in CNS [9,13,24], we decided to investigate oxidative stress in HC, PFC, and ST of mice treated with MCT. These brain areas were selected due to susceptibility to oxidative stress and they are involved with motor behavior.
Several studies have shown that ROS have an important role in the pathogenesis of many diseases, especially neurological and psychiatric diseases [36][37][38]. Oxidative stress may be a common pathogenic mechanism underlying many disorders in CNS, since the brain has comparatively greater vulnerability to oxidative damage [39]. The data presented in this study demonstrated that MCT (50 or 100 mg/kg) in the three brain areas increased TBARS, indicating an increase in lipid peroxidation. Membrane lipid derangements, including lipid peroxidation, have been reported to contribute significantly to paroxysmal membrane dysfunction in neurodegenerative diseases [40]. McEwen indicates that stressors were having neurotoxic effects on the hippocampus which predisposes to the development of depression [41]. Our results showed that the dose of MCT 50 or 100 mg/kg could contribute to alteration in CNS due to increased lipid peroxidation in the HP, PFC, and ST. In fact, besides the brain is more vulnerable to injury by lipid peroxidation products than other tissues, animals with high OS have an increase in prostaglandin levels and the overproduction of the compounds could release O 2 − and OH • [42]. Many studies have focused on the biochemical and molecular actions of nitric oxide (NO) in normal conditions, as well as its potential alteration in CNS [37,43,44]. We investigated MCT effects on nitrite levels and observed low levels of nitrite in all studied brain areas after MCT administration. The low response in the levels of nitrite/nitrate and in the values of HC, PFC, and ST may has occurred because the nitric oxide is an unstable molecule [45,46]. In the CNS, the major reactive nitrogen species (RNS) mediator of oxidative stress is peroxynitrite ONOO − [47]. The participation of peroxynitrite in oxidative stress is difficult to determine due to their reduced life span and interaction with other molecules [48]. However, it has been demonstrated that stressful conditions produce changes in NO metabolism and glutamatergic receptors to produce part of its stimulatory action on the CNS [49]. NO, in physiological levels, participates in a variety of physiological processes consisting of neurotransmission and regulation of blood vessel wall [50].
An elevation in free radical formation can be accompanied by an immediate compensatory increase in the activities of the free radical scavenging enzymes [51]. In our experimental situation, the catalase activity followed the increased levels of lipid peroxidation only in the HC. HC is extremely sensitive to the OS effects as compared to other brain areas [52]. The greater susceptibility of the HC to stress in comparison to the other tissues can be explained on the basis of the differential blood supply to these regions and it is more deeply situated in the diffusion of respiratory gases [53]. These results may represent a neuroprotective mechanism of catalase against the oxidative effects caused by MCT administration related to TBARS increase in the brain regions studied.
Oxidative stress has been related to many neurodegenerative diseases, such as ischemia and Parkinson disease [16]. Rotenone, a chemical that belongs to the family of isoflavones naturally found in the roots and stems of several plants, is used as a broad-spectrum pesticide [54]. It has characteristics alike MCT, such as potent inhibitor of mitochondrial complex-I and crosses the blood-brain barrier as well as the cell membrane easily because of its lipophilic structure [55]. The present results showed that MCT increases lipidic peroxidation and catalase activity, only in HC, while it decreases nitrite/nitrate contente. Hippocampus was probably more affected because it is a rich area in mitochondria. These are related to a production of antioxidant defense when the brain is attacked by a harmful agent as monocrotaline [56,57]. Rotenone, a neurotoxin used as a Parkinson model, also augments lipidic peroxidation and nitric oxide content in chronically treated rats [54]. This substance also increases catalase activity after chronic administration to rats [55].

Conclusion
Our findings showed that monocrotaline caused cell lesions in the HC and PFC regions. Also, it produced oxidative stress in the HC, PFC, and ST in mice. These alterations may contribute to the neurological effects related to this compound.