Sulforaphane protects from myocardial ischemia-reperfusion damage through the balanced activation of Nrf2/AhR.

The activation of the transcription factor Nrf2 and the consequent increment in the antioxidant response might be a powerful strategy to contend against reperfusion damage. In this study we compared the effectiveness between sulforaphane (SFN), a well known activator of Nrf2 and the mechanical maneuver of post-conditioning (PostC) to confer cardioprotection in an in vivo cardiac ischemia-reperfusion model. We also evaluated if additional mechanisms, besides Nrf2 activation contribute to cardioprotection. Our results showed that SFN exerts an enhanced protective response as compared to PostC. Bot, strategies preserved cardiac function, decreased infarct size, oxidative stress and inflammation, through common protective pathways; however, the aryl hydrocarbon receptor (AhR) also participated in the protection conferred by SFN. Our data suggest that SFN-mediated cardioprotection involves transient Nrf2 activation, followed by phase I enzymes upregulation at the end of reperfusion, as a long-term protection mechanism.

Senescence associated secretory phenotype profile from primary lung mice fibroblasts depends on the senescence induction stimuli.

Cellular senescence is a multifactorial phenomenon of growth arrest and distorted function, which has been recognized as an important feature during tumor suppression mechanisms and a contributor to aging. Senescent cells have an altered secretion pattern called Senescence-Associated Secretory Phenotype (SASP) that comprises a complex mix of factors including cytokines, growth factors, chemokines, and matrix metalloproteinases. SASP has been related with local inflammation that leads to cellular transformation and neurodegenerative diseases. Various pathways for senescence induction have been proposed; the most studied is replicative senescence due to telomere attrition called replicative senescence (RS). However, senescence can be prematurely achieved when cells are exposed to diverse stimuli such as oxidative stress (stress-induced premature senescence, SIPS) or proteasome inhibition (proteasome inhibition-induced premature senescence, PIIPS). SASP has been characterized in RS and SIPS but not in PIIPS. Hence, our aim was to determine SASP components in primary lung fibroblasts obtained from CD-1 mice induced to senescence by PIIPS and compare them to RS and SIPS. Our results showed important variations in the 62 cytokines analyzed, while SIPS and RS showed an increase in the secretion of most cytokines, and in PIIPS only 13 were incremented. Variations in glutathione-redox balance were also observed in SIPS and RS, and not in PIIPS. All senescence types SASP displayed a pro-inflammatory profile and increased proliferation in L929 mice fibroblasts exposed to SASP. However, the behavior observed was not exactly the same, suggesting that the senescence induction pathway might encompass dissimilar responses in adjacent cells and promote different outcomes.

Sulforaphane reduces the alterations induced by quinolinic acid: modulation of glutathione levels.

Glutamate-induced excitotoxicity involves a state of acute oxidative stress, which is a crucial event during neuronal degeneration and is part of the physiopathology of neurodegenerative diseases. In this work, we evaluated the ability of sulforaphane (SULF), a natural dietary isothiocyanate, to induce the activation of transcription factor Nrf2 (a master regulator of redox state in the cell) in a model of striatal degeneration in rats infused with quinolinic acid (QUIN). Male Wistar rats received SULF (5mg/kg, i.p.) 24h and 5min before the intrastriatal infusion of QUIN. SULF increased the reduced glutathione (GSH) levels 4h after QUIN infusion, which was associated with its ability to increase the activity of glutathione reductase (GR), an antioxidant enzyme capable to regenerate GSH levels at 24h. Moreover, SULF treatment increased glutathione peroxidase (GPx) activity, while no changes were observed in γ-glutamyl cysteine ligase (GCL) activity. SULF treatment also prevented QUIN-induced oxidative stress (measured by oxidized proteins levels), the histological damage and the circling behavior. These results suggest that the protective effect of SULF could be related to its ability to preserve GSH levels and increase GPx and GR activities.

NAD(P)H oxidase contributes to neurotoxicity in an excitotoxic/prooxidant model of Huntington's disease in rats: protective role of apocynin.

Intrastriatal injection of quinolinic acid (QUIN) to rodents reproduces some biochemical, morphological, and behavioral characteristics of Huntington's disease. NAD(P)H oxidase is an enzymatic complex that catalyzes superoxide anion (O(2).(-)) production from O(2) and NADPH. The present study evaluated the role of NAD(P)H oxidase in the striatal damage induced by QUIN (240 nmol/microl) in adult male Wistar rats by means of apocynin (APO; 5 mg/kg i.p.), a specific NAD(P)H oxidase inhibitor. Rats were given APO 30 min before and 1 hr after QUIN injection or only 30 min after QUIN injection. NAD(P)H oxidase activity was measured in striatal homogenates by O2(*)(-) production. QUIN infusion to rats significantly increased striatal NAD(P)H oxidase activity (2 hr postlesion), whereas APO treatments decreased the QUIN-induced enzyme activity (2 hr postlesion), lipid peroxidation (3 hr postlesion), circling behavior (6 days postlesion), and histological damage (7 days postlesion). The addition of NADH to striatal homogenates increased NAD(P)H oxidase activity in striata from QUIN-treated animals but not from sham rats. Interestingly, O2(*)(-) production in QUIN-lesioned striata was unaffected by the addition of substrates for intramitochondrial O2(*)(-) production, xanthine oxidase and nitric oxide synthase, suggesting that NAD(P)H oxidase may be the main source of O2(*)(-) in QUIN-treated rats. Moreover, the administration of MK-801 to rats as a pretreatment resulted in a complete prevention of the QUIN-induced NAD(P)H activation, suggesting that this toxic event is completely dependent on N-methyl-D-aspartate receptor overactivation. Our results also suggest that NAD(P)H oxidase is involved in the pathogenic events linked to excitotoxic/prooxidant conditions.

Excitotoxic brain damage involves early peroxynitrite formation in a model of Huntington's disease in rats: protective role of iron porphyrinate 5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinate iron (III).

Oxidative/nitrosative stress is involved in NMDA receptor-mediated excitotoxic brain damage produced by the glutamate analog quinolinic acid. The purpose of this work was to study a possible role of peroxynitrite, a reactive oxygen/nitrogen species, in the course of excitotoxic events evoked by quinolinic acid in the brain. The effects of Fe(TPPS) (5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinate iron (III)), an iron porphyrinate and putative peroxynitrite decomposition catalyst, were tested on lipid peroxidation and mitochondrial function in brain synaptic vesicles exposed to quinolinic acid, as well as on peroxynitrite formation, nitric oxide synthase and superoxide dismutase activities, lipid peroxidation, caspase-3-like activation, DNA fragmentation, and GABA levels in striatal tissue from rats lesioned by quinolinic acid. Circling behavior was also evaluated. Increasing concentrations of Fe(TPPS) reduced lipid peroxidation and mitochondrial dysfunction induced by quinolinic acid (100 microM) in synaptic vesicles in a concentration-dependent manner (10-800 microM). In addition, Fe(TPPS) (10 mg/kg, i.p.) administered 2 h before the striatal lesions, prevented the formation of peroxynitrite, the increased nitric oxide synthase activity, the decreased superoxide dismutase activity and the increased lipid peroxidation induced by quinolinic acid (240 nmol/microl) 120 min after the toxin infusion. Enhanced caspase-3-like activity and DNA fragmentation were also reduced by the porphyrinate 24 h after the injection of the excitotoxin. Circling behavior from quinolinic acid-treated rats was abolished by Fe(TPPS) six days after quinolinic acid injection, while the striatal levels of GABA, measured one day later, were partially recovered. The protective effects that Fe(TPPS) exerted on quinolinic acid-induced lipid peroxidation and mitochondrial dysfunction in synaptic vesicles suggest a primary action of the porphyrinate as an antioxidant molecule. In vivo findings suggest that the early production of peroxynitrite, altogether with the enhanced risk of superoxide anion (O2*-) and nitric oxide formation (its precursors) induced by quinolinic acid in the striatum, are attenuated by Fe(TPPS) through a recovery in the basal activities of nitric oxide synthase and superoxide dismutase. The porphyrinate-mediated reduction in DNA fragmentation simultaneous to the decrease in caspase-3-like activation from quinolinic acid-lesioned rats suggests a prevention in the risk of peroxynitrite-mediated apoptotic events during the course of excitotoxic damage in the striatum. In summary, the protective effects that Fe(TPPS) exhibited both under in vitro and in vivo conditions support an active role of peroxynitrite and its precursors in the pattern of brain damage elicited by excitotoxic events in the experimental model of Huntington's disease. The neuroprotective mechanisms of Fe(TPPS) are discussed.

In vivo hydroxyl radical formation after quinolinic acid infusion into rat corpus striatum.

We studied the effect of an acute infusion of quinolinic acid (QUIN) on in vivo hydroxyl radical (.OH) formation in the striatum of awake rats. Using the microdialysis technique, the generation of.OH was assessed through electrochemical detection of the salicylate hydroxylation product 2,3-dihydroxybenzoic acid (2,3-DHBA). The .OH extracellular levels increased up to 30 times over basal levels after QUIN infusion (240 nmol/microl), returning to the baseline 2 h later. This response was attenuated, but not abolished, by pretreatment with the NMDA receptor antagonist MK-801 (10 mg/kg, i.p.) 60 min before QUIN infusion. The mitochondrial toxin 3-nitropropionic acid (3-NPA, 500 nmol/microl) had stronger effects than QUIN on .OH generation, as well as on other markers of oxidative stress explored as potential consequences of .OH increased levels. These results support the hypothesis that early .OH generation contributes to the pattern of toxicity elicited by QUIN. The partial protection by MK-801 suggests that QUIN neurotoxicity is not completely explained through NMDA receptor overactivation, but it may also involve intrinsic QUIN oxidative properties.

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 microM). 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 microM) and APV (250 microM) on QUIN-induced LP were evaluated in synaptosomes 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.

Subchronic administration of sublethal doses of thallium to rats: effects on distribution and lipid peroxidation in brain regions.

Occupational exposure to thallium (Tl+) is known to be responsible for severe neurological manifestations in humans, including ataxia and paralysis; however, little is known yet about the precise mechanism of toxicity elicited by this heavy metal at sublethal doses and its brain distribution after chronic or subchronic exposures resulting from environmental contamination. In order to evaluate the levels of Tl in rat brain regions after a subchronic administration (30 days) of sublethal doses of Tl (I) acetate: 0.8 mg/kg (1/40 of LD(50)), 1.6 mg/kg (1/20 of LD(50)), we measured the concentrations of Tl by atomic absorption spectrophotometry. A possible role of oxidative injury in the pattern of toxicity exerted by Tl in the same brain regions, was also studied. Lipid peroxidation (LP) as a current marker of oxidative stress, was estimated by the generation of lipid fluorescent products. Higher concentrations of Tl were observed in brain tissue from adult rats treated with 1.6 mg/kg, as compared to those treated with 0.8 mg/kg. However, no differential distribution of Tl among regions was observed after administration of 0.8 mg/kg dose to rats, nor after 1. 6 mg/kg dose. We also found significant changes in LP both in corpus striatum and cerebellum from rats treated daily with 0.8 mg/kg Tl, whereas all regions from rats treated with 1.6 mg/kg Tl exhibited enhanced LP as compared to control. These findings suggest an active role of free radicals and oxidative events involved in the pattern of toxicity after exposure to sublethal doses of Tl, which are associated with regional susceptibility of the brain to this metal.

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.

Alveolar lesions induced by systemic administration of cocaine to rats.

In this work, alveolar lesions induced after systemic administration of cocaine (30 mg/kg per day, i.p.) to rats were evaluated both by light microscope analysis for morphological assessment as well as by measurement of the alveolar area as a quantitative index of the alveolar damage. Rats were examined after different times of exposure: 7, 15, 30, 45, 60 and 75 days. The histopathological evaluation of cocaine-treated rats revealed a remarkable thickening in some interalveolar septa, with interstitial hemorrhages, progressive thrombosis and transformation of reticular and elastic fibers into diffuse fibrosis. A significant decrease of the alveolar area was also observed. These findings are indicative of severe changes in capillaries, alveoli and bronchiole after cocaine exposure, which in turn may progressively disrupt the general function of the lungs. Differential mechanisms of systemic toxicity after cocaine exposure are discussed.

Dapsone prevents morphological lesions and lipid peroxidation induced by quinolinic acid in rat corpus striatum.

Increasing doses of dapsone were tested on rats administered intrastriatally with quinolinic acid in order to evaluate a possible protective action of this drug on the striatal lesions produced after the excessive activation of N-methyl-D-aspartate receptors. Morphological lesions were evaluated 7 days after the intrastriatal injection of quinolinate (240 nmol/microl) by light microscopy, and the ratio of neuronal damage per field was also estimated as a quantitative index of the striatal toxicity. Quinolinate alone produced extensive necrosis and loss of striatal neurons. No protective effects on the striatal tissue from quinolinate-treated rats were observed at lower doses of dapsone (6.25 and 9.375 mg/kg). However, at higher doses (12.5 and 25 mg/kg), dapsone prevented significantly the striatum from quinolinate toxicity. Dapsone alone had no effect on the striatal tissue from control rats. A single dose of dapsone (12.5 mg/kg) was tested also on the index of lipid peroxidation 2 h after the striatal injection of quinolinate, resulting in a significant protection (78% vs. QUIN). Findings of this study, in accordance with our previous reports, demonstrate the ability of dapsone to prevent the neuronal damage associated with the excitatory action of quinolinate via overactivation of NMDA receptors, and provide evidences to support the hypothesis that this drug is acting against the pattern of toxicity elicited by agonists of glutamate receptors.

Thallium toxicity.

Thallium (T1+) is a toxic heavy metal which was accidentally discovered by Sir William Crookes in 1861 by burning the dust from a sulfuric acid industrial plant. He observed a bright green spectral band that quickly disappeared. Crookes named the new element 'Thallium' (after thallos meaning young shoot). In 1862, Lamy described the same spectral line and studied both the physical and chemical properties of this new element (Prick, J.J.G., 1979. Thallium poisoning. In: Vinkrn, P.J., Bruyn, G.W. (Eds.), Intoxication of the Nervous System, Handbook of Clinical Neurology, vol. 36. North-Holland, New York. pp. 239-278).

Quinolinic acid neurotoxicity: in vivo increased copper and manganese content in rat corpus striatum after quinolinate intrastriatal injection.

Copper and manganese, two essential metals involved in physiological and physiopathological processes in the brain, were measured in corpora striata of rats 7 days after intrastriatal injection of quinolinic acid (QUIN, 240 nmol/l microliters), an N-methyl-D-aspartate (NMDA) receptor agonist with toxic activity. Seven days after QUIN administration, copper and manganese contents were assessed by graphite furnace atomic absorption spectrophotometry. Total copper content was increased by 152% in QUIN-treated rats (18.74 +/- 2.05 micrograms/g) as compared to control animals (7.44 +/- 1.15 micrograms/g), whereas manganese striatal levels were enhanced by 35% (0.30 +/- 0.02 microgram/g) vs. control values (0.22 +/- 0.02 microgram/g). Quinolinate-induced striatal increase in copper and manganese levels were prevented by 23% (9.18 +/- 1.43 micrograms/g) and -0.45% (0.22 +/- 0.03 microgram/g) vs. control values, respectively, in rats pretreated with an NMDA receptor antagonist, dizocilpine (MK-801, 10 mg/kg, i.p.), 60 min before QUIN administration. As an index of QUIN neurotoxicity, striatal GABA levels were also measured 7 days after QUIN injection. GABA content was decreased by-55% in QUIN-lesioned rats (96.37 +/- 8.92 micrograms/g), whereas MK-801 was able to block QUIN-induced GABA depletion by 2% (219.37 +/- 10.60) vs. control values (214.2 +/- 21.88 micrograms/g). These findings suggest that increased concentrations of transition metals can be mediated by selective overactivation of NMDA receptors and might be a consequence of neural loss as well as glial response to damage.

Experimental neuromyopathy induced by thallium in rats.

A histopathological study, with a light microscope, of experimental neuromyopathy produced by thallotoxicosis was undertaken in 40 newborn Wistar rats. Treatment consisted of a single i.p. injection of an aqueous solution of 16 mg kg-1 thallium(I) acetate 1 day after birth. Groups of ten animals were euthanized at either 8 or 50 days of age. Sural nerves, as well as peroneus muscle, were fixed in 10% formaldehyde solution for 15 days and then prepared for histopathological observation. At 8 days of age sural nerves of thallium-treated animals showed a moderate reduction in the large- and medium-sized fibres and several of the myelin sheaths had initial degeneration along the course of the axon. Interstitial oedema was found in both neural and muscular tissues. Distinct features of focal necrosis as well as small haemorrhages were seen in peroneus muscle. At 50 days of age, the lesions were more diffuse. Large and small myelinated fibres were found to be sinuous, fragmented and scanty. Alterations in the large- and medium-sized axons were seen and the myelin sheaths were altered along the course of the axon, suggesting a progressive distal axonopathy. Additionally, muscle fibres had myopathic changes with abnormal central nucleoli and the striated transverse fibres had disappeared in many areas of the sample. Several interstitial foci of muscular necrosis accompanied by phagocytosis and fibrosis were also present.

Changes in transition metal contents in rat brain regions after in vivo quinolinate intrastriatal administration.

Total copper and manganese contents were measured in five rat brain regions 7 days after a unilateral striatal injection of quinolinic acid (QUIN, 240 nmol/ 1 microliter), an endogenous N-methyl-D-aspartate (NMDA) receptor agonist. Concentrations of both transition metals were evaluated in tissue of brain cortex, hippocampus, corpus striatum, midbrain and cerebellum of saline- and QUIN-treated rats using graphite furnace atomic absorption spectrophotometry. Increases in copper content were observed after QUIN striatal injection in cerebellum, hippocampus, midbrain and corpus striatum (37, 55, 71 and 152% as compared against control values, respectively) but not in brain cortex. Manganese levels were found enhanced only in corpus striatum of QUIN-treated rats by 35% vs. control values, but not in all other brain regions analyzed. QUIN-induced increases in regional copper content were partially prevented in hippocampus, midbrain and striatum (17, 57, and 23% vs. control, respectively) by pretreatment of rats with an NMDA receptor antagonist, dizocilpine (MK-801, 10 mg/kg, i.p.), administered 60 min before QUIN microinjection. The same protective effect of dizocilpine was observed against QUIN-induced enhancement of striatal manganese content (-0.45% vs. control). These findings resemble those changes observed in postmortem Huntington's disease brains and suggest that alterations in regional content of copper, but not in manganese, may be a consequence of the spreading of QUIN-induced neurotoxic events into the striatal tissue to the neighboring regions of the brain, by action of QUIN on NMDA receptors.

Thallium increases monoamine oxidase activity and serotonin turnover rate in rat brain regions.

The effect of thallium acetate administration on monoaminergic pathways was studied in male Wistar rats using 30 mg/kg and 50 mg/kg acute IP doses. We found that thallium activated both monoamine oxidase (MAO) activity and serotonin turnover rate in rat brain regions, that may contribute to the neuronal damage mechanism of the agent. MAO activity in midbrain and pons was increased at both doses (at 30 mg/kg dose by 27.7% and 37%; at 50 mg/kg dose by 48% and 47%, respectively vs. control group). Serotonin turnover rate in pons was also increased at the 30 mg/kg dose (172%) while midbrain and pons serotonin turnover was increased only at the 50 mg/kg dose (56% and 166%, respectively vs. control group). Dopamine turnover rate was not significantly changed. The results indicate that thallium induced a significant increase in pons and midbrain MAO activity and also in serotonin turnover rate as compared with control animals, and this could led to behavioral and toxic alterations in the rats intoxicated with thallium.

Thallium distribution in organs and brain regions of developing rats.

The concentration of thallium in body organs and brain regions was studied in rats as a function of the animals age from newborn to 20-days old. Thallium was analyzed at different times after a single sublethal i.p. injection of the metal (16 mg/kg). The results indicate that the brain is less permeable to thallium in the older animals, suggesting that reduced thallium transport into the brain is related to the establishment of the blood-brain barrier in the rats. Differences between weanling and newborn rats were also found in regard to regional distribution of thallium in the brain as the older animals showed a region-dependent distribution while newborn rats presented an homogeneous content of thallium among all regions.

Osteochondral lesions in developing rats intoxicated with thallium twenty four hours after birth.

An i.p. injection of a solution of thallium acetate in deionized water at a dose of 32 mg/kg, in 24-h-old rats, produces morphological and biochemical alterations in both cartilaginous and osseous tissues. From the beginning, there are alterations in the cartilaginous cell as well as in chrondrine, osteoblasts, osseous tissue and bone marrow. Rats were sacrificed at 24, 48, and 72 h and also at 7 days. Two animals survived for 50 days. One showed total irreversible alopecia while the other one had partial alopecia with discrete recovery. Both showed a low weight and a size of 8 cm. Microscopically, degenerative changes were produced consisting of alteration and death of many cartilaginous cells, uneven metachromasia and the chondrine and decrease of the growth cartilage, scanty bone trabeculae with few osteoblasts. The bone marrow showed few myeloblasts and megakaryocytes. Progressive cellular damage throughout the 50 days of survival represents a response of the thallium ionic accumulation and recycling in cellular mitochondria of all the body's cells. This appeared in our study as irreversible and progressive osteochondral alterations with atrophy of the skin and its adnexa, hyalinization of elastic and collagenous fibers with intense interstitial edema.

Neurotoxicity of thallium: biochemical and morphological study of organic lesions.

A biochemical study of experimental thallium intoxication in newborn rats and its morphological consequences upon the brain of developing animals was undertaken. The thallium content was analyzed in the following encephalic regions: hippocampus, hypothalamus, mesencephalon, cerebellum, and cortex. One day after application of the toxic substance, a homogeneous distribution of the metal in the brain was found. Thallium concentrations in the mentioned regions were twice as high as those found in an adult rat, at the same dosage, 20 newborn Wistar rats were used for the histopathological study. 5 remained as witnesses, the rest received a single injection of thallium of .07 ml. of a solution with a concentration of .32 mg/Kg. 3 rats were sacrificed at 24, 48 and 72 hours; 3 at 7 days and 3 at 51 days. The brain , sciatic and crural nerves were fixated in 10% formaldehyde for 15 days. Cuts in paraffin and frozen sections measuring between 5 and 7 microns were taken from the fragments of different areas. These were stained with aniline methods (Masson, Gallego and H-E) and silver-gold impregnation as modified by Río-Hortega.