Abel Santamaría

S-Allylcysteine, a garlic-derived antioxidant, ameliorates quinolinic acid-induced neurotoxicity and oxidative damage in rats.

Excitotoxicity elicited by overactivation of N-methyl-D-aspartate receptors is a well-known characteristic of quinolinic acid-induced neurotoxicity. However, since many experimental evidences suggest that the actions of quinolinic acid also involve reactive oxygen species formation and oxidative stress as major features of its pattern of toxicity, the use of antioxidants as experimental tools against the deleterious effects evoked by this neurotoxin becomes more relevant. In this work, we investigated the effect of a garlic-derived compound and well-characterized free radical scavenger, S-allylcysteine, on quinolinic acid-induced striatal neurotoxicity and oxidative damage. For this purpose, rats were administered S-allylcysteine (150, 300 or 450 mg/kg, i.p.) 30 min before a single striatal infusion of 1 microl of quinolinic acid (240 nmol). The lower dose (150 mg/kg) of S-allylcysteine resulted effective to prevent only the quinolinate-induced lipid peroxidation (P < 0.05), whereas the systemic administration of 300 mg/kg of this compound to rats decreased effectively the quinolinic acid-induced oxidative injury measured as striatal reactive oxygen species formation (P < 0.01) and lipid peroxidation (P < 0.05). S-Allylcysteine (300 mg/kg) also prevented the striatal decrease of copper/zinc-superoxide dismutase activity (P < 0.05) produced by quinolinate. In addition, S-allylcysteine, at the same dose tested, was able to reduce the quinolinic acid-induced neurotoxicity evaluated as circling behavior (P < 0.01) and striatal morphologic alterations. In summary, S-allylcysteine ameliorates the in vivo quinolinate striatal toxicity by a mechanism related to its ability to: (a) scavenge free radicals; (b) decrease oxidative stress; and (c) preserve the striatal activity of Cu,Zn-superoxide dismutase (Cu,Zn-SOD). This antioxidant effect seems to be responsible for the preservation of the morphological and functional integrity of the striatum.

MK-801, an N-methyl-d-aspartate receptor antagonist, blocks quinolinic acid-induced lipid peroxidation in rat corpus striatum

In this study, we evaluate the possible participation of lipid peroxidation (LP) in the neurotoxic events that follow after quinolinic acid (QUIN) microinjection into the rat corpus striatum. Two hours after QUIN (240 nmol/microliters) intrastriatal administration, lipid peroxidation was found increased by 32% vs. control as measured by thiobarbituric acid-reactive substances (TBARS). At the same time tested, the enhancement in LP was of 55% vs. control as measured by lipid fluorescent products (LFP) formation (a second index of lipid peroxidation employed). The increase of QUIN-induced lipid peroxidation was completely abolished by pretreatment of rats with an N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (10 mg/kg, i.p.), 60 min before QUIN microinjection. Results suggest an NMDA receptor involvement in the QUIN-induced oxidative processes.

Excitotoxic damage, disrupted energy metabolism, and oxidative stress in the rat brain: antioxidant and neuroprotective effects of L-carnitine

Excitotoxicity and disrupted energy metabolism are major events leading to nerve cell death in neurodegenerative disorders. These cooperative pathways share one common aspect: triggering of oxidative stress by free radical formation. In this work, we evaluated the effects of the antioxidant and energy precursor, levocarnitine (l‐CAR), on the oxidative damage and the behavioral, morphological, and neurochemical alterations produced in nerve tissue by the excitotoxin and free radical precursor, quinolinic acid (2,3‐pyrindin dicarboxylic acid; QUIN), and the mitochondrial toxin, 3‐nitropropionic acid (3‐NP). Oxidative damage was assessed by the estimation of reactive oxygen species formation, lipid peroxidation, and mitochondrial dysfunction in synaptosomal fractions. Behavioral, morphological, and neurochemical alterations were evaluated as markers of neurotoxicity in animals systemically administered with l‐CAR, chronically injected with 3‐NP and/or intrastriatally infused with QUIN. At micromolar concentrations, l‐CAR reduced the three markers of oxidative stress stimulated by both toxins alone or in combination. l‐CAR also prevented the rotation behavior evoked by QUIN and the hypokinetic pattern induced by 3‐NP in rats. Morphological alterations produced by both toxins (increased striatal glial fibrillary acidic protein‐immunoreactivity for QUIN and enhanced neuronal damage in different brain regions for 3‐NP) were reduced by l‐CAR. In addition, l‐CAR prevented the synergistic action of 3‐NP and QUIN to increase motor asymmetry and depleted striatal GABA levels. Our results suggest that the protective properties of l‐CAR in the neurotoxic models tested are mostly mediated by its characteristics as an antioxidant agent.

Effect of quinolinic acid on endogenous antioxidants in rat corpus striatum

The response of endogenous antioxidants to the N-methyl-D-aspartate (NMDA) receptor agonist and excitotoxin, quinolinic acid (QUIN), was investigated in rat corpus striatum. Animals treated with QUIN (240 nmol/microl), were sacrificed at 120 min after a single intrastriatal injection to examine the alterations in the levels of both reduced (GSH) and oxidized (GSSG) glutathione, and the activities of the antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (Gpx). Changes in the rate of lipid peroxidation (LP) were also measured after exposure to different doses of QUIN (60, 120, 240 and 480 nmol/microl) as an index of oxidative stress. When compared to control, lipid peroxidation was increased at QUIN doses of 240 and 480 nmol/microl. Striatal levels of GSH and GSSG were decreased and increased, respectively, after QUIN injection; whereas GPx activity was unchanged. Cytosolic copper/zinc SOD (CuZn-SOD) activity decreased after treatment, while mitochondrial manganese SOD (Mn-SOD) was unchanged. The alterations observed on these antioxidant systems suggest that QUIN toxicity is mediated by specific mechanisms leading to oxidative stress.

Quinolinic acid induces oxidative stress in rat brain synaptosomes

The oxidative action of quinolinic acid (QUIN), and the protective effects of glutathione (GSH), and 2-amino-5-phosphonovaleric acid (APV), were tested in rat brain synaptosomes, Reactive oxygen species (ROS) formation was quantified after the exposure of synaptosomes to increasing concentrations of QUIN (25–500 μM). The potency of QUIN to induce lipid peroxidation (LP) was tested as a regional index of thiobarbituric acid-reactive substances (TBARS) production, and the antioxidant actions of both GSH (50 μM) and APV (250 μM) on QUIN-induced LP were evaluated in synapto somes prepared from different brain regions. QUIN induced concentration-dependent increases in ROS formation and TBARS in all regions analyzed, but increased production of fluorescent peroxidized lipids only in the striatum and the hippocampus, whereas both GSH and APV decreased this index. These results suggest that the excitotoxic action of QUIN involves regional selectivity in the oxidative status of brain synaptosomes, and may be prevented by substances exhibiting antagonism at the NMDA receptor.

Protective effects of the antioxidant selenium on quinolinic acid-induced neurotoxicity in rats: in vitro and in vivo studies

Quinolinic acid (QUIN), a well known excitotoxin that produces a pharmacological model of Huntingtons disease in rats and primates, has been shown to evoke degenerative events in nerve tissue via NMDA receptor (NMDAr) overactivation and oxidative stress. In this study, the antioxidant selenium (as sodium selenite) was tested against different markers of QUIN‐induced neurotoxicity under both in vitro and in vivo conditions. In the in vitro experiments, a concentration‐dependent effect of selenium was evaluated on the regional peroxidative action of QUIN as an index of oxidative toxicity in rat brain synaptosomes. In the in vivo experiments, selenium (0.625 mg per kg per day, i.p.) was administered to rats for 5 days, and 2 h later animals received a single unilateral striatal injection of QUIN (240 nmol/µL). Rats were killed 2 h after the induction of lesions with QUIN to measure lipid peroxidation and glutathione peroxidase (GPx) activity in striatal tissue. In other groups, the rotation behavior, GABA content, morphologic alterations, and the corresponding ratio of neuronal damage were all evaluated as additional markers of QUIN‐induced striatal toxicity 7 days after the intrastriatal injection of QUIN. Selenium decreased the peroxidative action of QUIN in synaptosomes both from whole rat brain and from the striatum and hippocampus, but not in the cortex. A protective concentration‐dependent effect of selenium was observed in QUIN‐exposed synaptosomes from whole brain and hippocampus. Selenium pre‐treatment decreased the in vivo lipid peroxidation and increased the GPx activity in QUIN‐treated rats. Selenium also significantly attenuated the QUIN‐induced circling behavior, the striatal GABA depletion, the ratio of neuronal damage, and partially prevented the morphologic alterations in rats. These data suggest that major features of QUIN‐induced neurotoxicity are partially mediated by free radical formation and oxidative stress, and that selenium partially protects against QUIN toxicity.

3-Nitropropionic acid as a tool to study the mechanisms involved in Huntington's disease: past, present and future.

Huntington’s disease (HD) is an inheritable autosomal-dominant disorder whose causal mechanisms remain unknown. Experimental models have begun to uncover these pathways, thus helping to understand the mechanisms implicated and allowing for the characterization of potential targets for new therapeutic strategies. 3-Nitropropionic acid is known to produce in animals behavioural, biochemical and morphologic changes similar to those occurring in HD. For this reason, this phenotypic model is gaining attention as a valuable tool to mimick this disorder and further developing new therapies. In this review, we will focus on the past and present research of this molecule, to finally bring a perspective on what will be next in this promising field of study.

The Antioxidant Mechanisms Underlying the Aged Garlic Extract- and S-Allylcysteine-Induced Protection

Aged garlic extract (AGE) is an odorless garlic preparation containing S-allylcysteine (SAC) as its most abundant compound. A large number of studies have demonstrated the antioxidant activity of AGE and SAC in both in vivo—in diverse experimental animal models associated to oxidative stress—and in vitro conditions—using several methods to scavenge reactive oxygen species or to induce oxidative damage. Derived from these experiments, the protective effects of AGE and SAC have been associated with the prevention or amelioration of oxidative stress. In this work, we reviewed different antioxidant mechanisms (scavenging of free radicals and prooxidant species, induction of antioxidant enzymes, activation of Nrf2 factor, inhibition of prooxidant enzymes, and chelating effects) involved in the protective actions of AGE and SAC, thereby emphasizing their potential use as therapeutic agents. In addition, we highlight the ability of SAC to activate Nrf2 factor—a master regulator of the cellular redox state. Here, we include original data showing the ability of SAC to activate Nrf2 factor in cerebral cortex. Therefore, we conclude that the therapeutic properties of these molecules comprise cellular and molecular mechanisms at different levels.

Homocysteine-induced brain lipid peroxidation: Effects of NMDA receptor blockade, antioxidant treatment, and nitric oxide synthase inhibition

The effect of homocysteine (HCY) on lipid peroxidation (LP), a current mechanism of oxidative neurotoxicity, was investigated in rat brain synaptosomes. LP was assessed by measuring the amount of thiobarbituric acid-reactive substances (TBARS) formed from synaptosomal fractions following HCY treatment. Increasing HCY concentrations (5–1000 μM) enhanced the TBARS formation in brain synaptosomes in a concentration-dependent manner. When compared at equimolar concentrations (100 μM), the oxidative potency of HCY was lower than that of the oxidant ferrous sulfate, similar to that produced by glutamate (Glu) and the mitochondrial toxin 3-nitropropionic acid, and higher than that of the Glu agonists, kainate and quinolinate. TheN-methyl-D-aspartate receptor (NMDAr) antagonist dizocilpine (MK-801) completely blocked the HCY-induced LP at concentrations from 5 to 1000 μM, whereas the well-known antioxidantN-acetylcysteine (NAC) was less effective, but still protective against the HCY oxidative toxicity at higher concentrations (400 and 1000 μM). Three nitric oxide synthase (NOS) inhibitors, 7-nitroindazole (7-NI),Nω-nitro-L-arginine (L-NARG) andNω-nitro-L-arginine methyl ester (L-NAME), were also tested on HCY-induced LP at increasing concentrations. Both nonspecific NOS effectively the HCY-induced LP than did the selective neuronal NOS inhibitor, 7-NI. These results show that submillimolar concentrations of HCY can induce oxidative injury to nerve terminals, and this effect involves NMDAr stimulation, NOS activation, and associated free radicals formation.

Increased Formation of Reactive Oxygen Species, but No Changes in Glutathione Peroxidase Activity, in Striata of Mice Transgenic for the Huntington's Disease Mutation

The formation of reactive oxygen species (ROS) and the activities of the antioxidant enzymes glutathione peroxidase (GPx) and catalase (CAT) were measured as a function of age in the striatum of mice transgenic for the Huntingtons disease (HD) mutation. Striata from R6/1 transgenic male mice were dissected at different ages (11, 19, and 35 weeks). The amount of dichlorofluorescein (DCF), an index of ROS formation, was significantly increased in R6/1 mice at all ages tested, whereas GPx activity remained unchanged when compared with wild-type control animals in all groups evaluated. CAT activity was very low, just above detection in the striata of both control and transgenic mice. Nineteen and 35-week-old R6/1 mice also developed feet clasping behavior, but only 35-week-old animals showed body weight loss. Our findings support an active role of free radicals in the onset and progression of the neurological phenotype of R6/1 mice. We suggest that changes in ROS formation are due to an age-related increased propensity of the striatum of transgenic animals to generate oxygen radicals as a response to the evolving pathological conditions.