Rafael Porto Ineu

Diphenyl diselenide reverses gastric lesions in rats: Involvement of oxidative stress

The aim of the present study was to evaluate if diphenyl diselenide administered by oral route was effective in restoring gastric lesions induced by ethanol. The possible involvement of oxidative stress in diphenyl diselenide antiulcer effect was also evaluated. Different doses of diphenyl diselenide (dissolved in soya bean oil, 1mL/kg) were administered orally 1h before (pre-treatment study) or 1h after ethanol (70%, v/v, 2mL/kg, post-treatment study). Ulcer lesions were quantified 1h after ethanol administration (pre-treatment protocol) or 1h after diphenyl diselenide study (post-treatment protocol). Diphenyl diselenide (0.1-10mg/kg or 0.32-32micromol/kg), when administered previously or posteriorly prevented and reversed respectively, the development of gastric lesions induced by ethanol in rats. A number of markers of oxidative stress were examined in rat stomach including thiobarbituric acid reactive species (TBARS), catalase (CAT), superoxide dismutase (SOD), non-protein thiol groups (NPSH) and ascorbic acid. In addition to attenuating the gastric lesions, low doses of diphenyl diselenide prevented (pre-treatment) or reversed (post-treatment) the ethanol-induced changes in TBARS, SOD activity and ascorbic acid content. In conclusion, the present data reveal that diphenyl diselenide, administered by oral route, possesses an antiulcer effect by modulating antioxidant mechanisms.

Transient receptor potential ankyrin 1 receptor stimulation by hydrogen peroxide is critical to trigger pain during monosodium urate-induced inflammation in rodents.

OBJECTIVE Gout is a common cause of inflammatory arthritis and is provoked by the accumulation of monosodium urate (MSU) crystals. However, the underlying mechanisms of the pain associated with acute attacks of gout are poorly understood. The aim of this study was to evaluate the role of transient receptor potential ankyrin 1 (TRPA-1) and TRPA-1 stimulants, such as H2 O2 , in a rodent model of MSU-induced inflammation. METHODS MSU or H2 O2 was injected into the hind paws of rodents or applied in cultured sensory neurons, and the intracellular calcium response was measured in vitro. Inflammatory or nociceptive responses in vivo were evaluated using pharmacologic, genetic, or biochemical tools and methods. RESULTS TRPA-1 antagonism, TRPA-1 gene deletion, or pretreatment of peptidergic TRP-expressing primary sensory neurons with capsaicin markedly decreased MSU-induced nociception and edema. In addition to these neurogenic effects, MSU increased H2 O2 levels in the injected tissue, an effect that was abolished by the H2 O2 -detoxifying enzyme catalase. H2 O2 , but not MSU, directly stimulated sensory neurons through the activation of TRPA-1. The nociceptive responses evoked by MSU or H2 O2 injection were attenuated by the reducing agent dithiothreitol. In addition, MSU injection increased the expression of TRPA-1 and TRP vanilloid channel 1 (TRPV-1) and also enhanced cellular infiltration and interleukin-1β levels, and these effects were blocked by TRPA-1 antagonism. CONCLUSION Our results suggest that MSU injection increases tissue H2 O2 , thereby stimulating TRPA-1 on sensory nerve endings to produce inflammation and nociception. TRPV-1, by a previously unknown mechanism, also contributes to these responses.

Delayed biochemical changes induced by mercury intoxication are prevented by zinc pre-exposure.

This work evaluated the delayed effects of mercury and the effectiveness of zinc in preventing such effects. Pups were pre-treated with 1 daily dose of ZnCl(2) (27 mg/kg/day, by subcutaneous injections) from 3rd to 7th postnatal day and received 1 daily dose of 5 mg/kg of HgCl(2), for 5 subsequent days (8-12 days old). Animals were euthanized 21 days after the end of Hg-exposure. Porphobilinogen-synthase activity as well as zinc and mercury contents was determined in the liver and kidneys. Alanine aminotransferase, aspartate aminotransferase and lactic dehydrogenase activities as well as urea, creatinine and glucose levels were analyzed in plasma or serum. Some animals were considered more sensitive to mercury, since they did not recover the body weight gain and presented an increase of renal and hepatic mercury content, urea and creatinine levels; a decrease in renal porphobilinogen-synthase and alanine aminotransferase activities, as well as a decrease in the liver and an increase in kidney weights. Some animals were considered less sensitive to mercury because they recovered the body weight and presented no biochemical alterations in spite of mercury in the tissues. Zinc prevents partially or totally the alterations caused by mercury even those that persisted for a long time after the end of exposure. These findings suggest that there is difference among the animals regarding the sensitivity to mercury.

Complex methylmercury-cysteine alters mercury accumulation in different tissues of mice

Methylmercury (MeHg) can cause deleterious effects in vertebrate tissues, particularly in the central nervous system. MeHg interacts with sulfhydryl groups from low and high molecular weight thiols in the blood, which can facilitate MeHg uptake into different tissues. The purpose of this study was to examine the effect of MeHg-Cysteine (MeHg-Cys) complex administration on Hg-uptake in cerebral areas (cortex and cerebellum), liver and kidney of adult mice. Animals were divided into four groups: control (1 mL/kg distilled water), MeHg (2 mg/kg), Cys (2 mg/kg) and MeHg-Cys complex (0.8 molar ratio). Mice received one intraperitoneal injection per day for 60 consecutive days. Treatment with MeHg significantly increased mercury concentrations in all tissues analysed when compared with the control group. The accumulation of mercury in brain and in liver was further increased in animals that received MeHg-Cys complex when compared with the MeHg alone group. However, renal Hg decreased in MeHg-Cys treated mice, when compared with the group treated only with MeHg. In summary, the transport of MeHg-Cys complex was tissue-specific, and we observed an increase in its uptake by liver and brain as well as a decrease in kidney.

Antioxidant effect of zinc chloride against ethanol-induced gastrointestinal lesions in rats

The aim of the present study was to evaluate the possible effects of zinc chloride against the gastrointestinal lesions caused by oral administration of ethanol in rats. Rats were divided into five groups, namely, saline, ethanol, zn, zn+ethanol and ethanol+zn. Ethanol 70% (2 mL/kg) was administered by gavage in 36 h fasted rats. Zinc chloride (27 mg/kg, ~13 mg/kg of zinc) was given by gavage 1h before or 1h after the administration of ethanol. Oral administration of ethanol consistently induced damage in the rat glandular stomach and intestine. Zinc did not demonstrate effect per se and significantly reduced gastrointestinal lesions when administered either before or after lesion induction. Ethanol induced enhancement of thiobarbituric acid reactive substance and reactive species levels, diminished the ascorbic acid and total protein SH content as well as superoxide dismutase and catalase activity in stomach and intestine of rats. Zinc treatment prevented and reversed these alterations induced by ethanol. Stomach and intestine of rats treated with zinc presented higher zinc content than the tissues of rats treated only with ethanol. Non-protein SH content was not altered by any treatment. Results suggested that the gastrointestinal protective effect of zinc in this experimental model could be due to its antioxidant effect.

Lactating and non-lactating rats differ in sensitivity to HgCl2: Protective effect of ZnCl2

This work investigated zinc (Zn) and mercury (Hg) effects on oxidative parameters, markers of toxicity and metal levels in different tissues from non-lactating rats (NLR) and lactating rats (LR). Adult NLR and LR received ZnCl2 (27mg/kg) or saline (0.9%) subcutaneously and after 24h they received HgCl2 (5mg/kg) or saline (0.9%). Twenty four hours later, they were sacrificed and the preparation of biological material and biochemical analyses were performed. With respect to oxidative parameters, Hg exposure decreased kidney total SH levels from NLR and LR and hepatic catalase activity (not statistically significant) in NLR. Zinc pre-treatment partly prevented the decrease of kidney total SH levels in LR. Zinc per se increased hepatic non-protein SH levels of NLR and LR. Regarding toxicity markers, Hg exposure inhibited the δ-aminolevulinic acid dehydratase (δ-ALA-D) activity from kidney and liver of NLR, inhibited serum alanine aminotransferase (ALT) activity of LR and increased serum creatinine and urea levels of NLR and LR. Zinc pre-exposure prevented the enzymatic alterations caused by Hg. NLR and LR Hg exposed presented accumulation of mercury in the kidney, liver, blood and urine. Zinc pre-treatment prevented this accumulation partly in NLR liver and blood and completely in LR kidney and liver. These results show that NLR and LR are differently sensitive to HgCl2 and that ZnCl2 showed a promising effect against Hg toxicity.

Biochemical parameters of pregnant rats and their offspring exposed to different doses of inorganic mercury in drinking water.

This work investigated the effects of low and high doses of inorganic mercury in drinking water on biochemical parameters of pregnant rats and their offspring. Female Wistar rats were treated during pregnancy with 0, 0.2, 0.5, 10 or 50 μg Hg(2+)/mL as HgCl(2). Rats were euthanized on day 20 of pregnancy. Pregnant rats presented a decrease in total water intake in all doses of mercury tested. At high doses, a decrease in the total food intake and in body weight gain was observed. Pregnant rats exposed to 50 μg Hg(2+)/mL presented an increase in kidney relative weight. Mercury exposure did not change serum urea and creatinine levels in any of the doses tested. Moreover, mercury exposure did not change porphobilinogen synthase activity of kidney, liver and placenta from pregnant rats in any of the doses tested, whereas fetuses of pregnant rats exposed to 50 μg Hg(2+)/mL presented an increase in the hepatic porphobilinogen synthase activity. In general, pregnant rats presented alterations due to HgCl(2) exposure in drinking water. However, only the dose 50 μg Hg(2+)/mL appeared to be enough to cross the blood-placenta barrier, since at this dose the fetuses presented change in the porphobilinogen synthase activity.

Preventive Effect of CuCl2 on Behavioral Alterations and Mercury Accumulation in Central Nervous System Induced by HgCl2 in Newborn Rats

This study investigated the benefits of Cu preexposition on Hg effects on behavioral tests, acetylcholinesterase (AChE) activity and Hg, and essential metal contents in the cerebrum and cerebellum of neonate rats. Wistar rats received (subcutaneous) saline or CuCl2·2H2O (6.9 mg/kg/day) when they were 3 to 7 days old and saline or HgCl2 (5.0 mg/kg/day) when they were 8 to 12 days old. Mercury exposure reduced the performance of rats in the negative geotaxis (3–13 days) and beaker test (17–20 days), inhibited cerebellum AChE activity (13 days), increased cerebrum and cerebellum Hg (13 days), cerebrum Cu (13 days), and cerebrum and cerebellum Zn levels (33 days). The performance of rats in the tail immersion and rotarod tests as well as Fe and Mg levels were not altered by treatments. Copper prevented all alterations induced by mercury. These results are important to open a new perspective of prevention and/or therapy for mercury exposure.

Antioxidant and antiulcer potential of aqueous leaf extract of Kigelia africana against ethanol-induced ulcer in rats

Ethnobotanical claims regarding Kigelia africana reported antiulcer properties as part of its medicinal application. In this work, aqueous leaf extract from K. africana was investigated for its phytochemical constituents and antiulcer potential against ethanol-induced ulcer in rats. The participation of oxidative stress on ethanol-induced ulcer and the potential protective antioxidant activity of K. africana extracts were investigated by determining vitamin C and thiobarbituric acid reactive species (TBARS) contents in the gastric mucosa of rats. The HPLC analysis showed the presence of gallic acid, chlorogenic acid, caffeic acid and also the flavonoids rutin, quercetin and kaempferol in the aqueous plant extract. Oral treatment with K. africana extract (1.75; 3.5; 7 and 14 mg/kg) one hour after ulcer induction with ethanol decreased in a dose dependent manner the ulcer index. Ethanol increased significantly stomachal TBARS levels and decreased vitamin C content when compared to the control animals. K. africana blunted the ethanol-induced oxidative stress and restored vitamin C content to the control levels. The present results indicate that the aqueous leaf extract from K. africana possesses antiulcer potential. The presence of flavonoids in plant extract suggests that its antiulcerogenic potential is associated with antioxidant activity. Of particular therapeutic potential, K. africana was effective against ethanol even after the induction of ulcer, indicating that it can have protective and curative effects against gastric lesion.

PhKv a toxin isolated from the spider venom induces antinociception by inhibition of cholinesterase activating cholinergic system

Abstract Background and aims Cholinergic agents cause antinociception by mimicking the release of acetylcholine (ACh) from spinal cholinergic nerves. PhKv is a peptide isolated from the venom of the armed spider Phoneutria nigriventer. It has an antiarrythmogenic activity that involves the enhanced release of acetylcholine. The aim of this study was to investigate whether PhKv had an antinociceptive action in mice. Methods Male albino Swiss mice (25–35 g) were used in this study. The PhKv toxin was purified from a PhTx3 fraction of the Phoneutria nigriventer spider’s venom. Because of its peptide nature, PhKv is not orally available and it was delivered directly into the central nervous system by an intrathecal (i.t.) route. PhKV on the thermal and mechanical sensitivity was evaluated using plantar test apparatus and the up-and-down method. The analgesic effects of PhKv were studied in neuropathic pain (CCI) and in the peripheral capsicin test. In order to test whether PhKv interfered with the cholinergic system, the mice were pre-treated with atropine (5mg/kg, i.p.) or mecamylamine (0.001 mg/kg, i.p.) and the PhKv toxin (30 pmol/site i.t.) or neostigmine (100 pmol/site) were applied 15 min before the intraplantar capsaicin (1 nmol/paw) administrations. To investigate PhKv action on the AChE activities, was performed in vitro and ex vivo assay for AChE. For the in vitro experiments, mice spinal cord supernatants of tissue homogenates (1 mg/ml) were used as source of AChE activity. The AChE assay was monitored at 37 °C for 10 min in a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) at 405 nm. Results PhKv (30 and 100pmol/site, i.t.) had no effect on the thermal or mechanical sensitivity thresholds. However, in a chronic constriction injury model of pain, PhKv (10pmol/site, i.t.) caused a robust reduction in mechanical withdrawal with an antinociceptive effect that lasted 4 h. A pretreatment in mice with PhKv (30pmol/site, i.t.) or neostigmine (100pmol/site, i.t.) 15min before an intraplantar injection of capsaicin (1 nmol/paw) caused a maximal antinociceptive effect of 69.5 ± 4.9% and 85 ± 2.5%, respectively. A pretreatment in mice with atropine; 5 mg/kg, i.p. or mecamylamine 0.001 mg/kg, i.p. inhibited a neostigimine and PhKv-induced antinociception, suggesting a cholinergic mechanism. Spinal acetylcholinesterase was inhibited by PhKv with ED50 of 7.6 (4.6–12.6 pmol/site, i.t.). PhKv also inhibited the in vitro AChE activity of spinal cord homogenates with an EC50 of 20.8 (11.6–37.3 nM), shifting the Km value from 0.06 mM to 18.5 mM, characterizing a competitive inhibition of AChE activity by PhKv. Conclusions Our findings provide, to our knowledge, the first evidence that PhKv caused inhibition of AChE, it increased the ACh content at the neuronal synapses, leading to an activation of the cholinergic system and an antinociceptive response. Implications Studies regarding the nociceptive mechanisms and the identification of potential targets for the treatment of pain have become top priorities. PhKv, by its action of stimulating the cholinergic receptors muscarinic and nicotinic system, reduces pain it may be an alternative for controlling the pain processes.