Tactile Stimulation on Adulthood Modifies the HPA Axis, Neurotrophic Factors, and GFAP Signaling Reverting Depression-Like Behavior in Female Rats.

Depression is a common psychiatric disease which pharmacological treatment relieves symptoms, but still far from ideal. Tactile stimulation (TS) has shown beneficial influences in neuropsychiatric disorders, but the mechanism of action is not clear. Here, we evaluated the TS influence when applied on adult female rats previously exposed to a reserpine-induced depression-like animal model. Immediately after reserpine model (1 mg/kg/mL, 1×/day, for 3 days), female Wistar rats were submitted to TS (15 min, 3×/day, for 8 days) or not (unhandled). Imipramine (10 mg/kg/mL) was used as positive control. After behavioral assessments, animals were euthanized to collect plasma and prefrontal cortex (PFC). Behavioral observations in the forced swimming test, splash test, and sucrose preference confirmed the reserpine-induced depression-like behavior, which was reversed by TS. Our findings showed that reserpine increased plasma levels of adrenocorticotropic hormone and corticosterone, decreased brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B, and increased proBDNF immunoreactivity in the PFC, which were also reversed by TS. Moreover, TS reestablished glial fibrillary acidic protein and glucocorticoid receptor levels, decreased by reserpine in PFC, while glial cell line-derived neurotrophic factor was increased by TS per se. Our outcomes are showing that TS applied in adulthood exerts a beneficial influence in depression-like behaviors, modulating the HPA axis and regulating neurotrophic factors more effectively than imipramine. Based on this, our proposal is that TS, in the long term, could be considered a new therapeutic strategy for neuropsychiatric disorders improvement in adult life, which may represent an interesting contribution to conventional pharmacological treatment.

Fluconazole induces genotoxicity in cultured human peripheral blood mononuclear cells via immunomodulation of TNF-α, IL-6, and IL-10: new challenges for safe therapeutic regimens.

Context: Fluconazole (FNZ) is a drug used in antifungal therapy. However, the minimum FNZ dose to interfering with immune responses or inducing DNA damage is still unknown. Objective: This study investigated the toxicological profile of FNZ on cultured human peripheral blood mononuclear cells (PBMCs) treated with different concentrations of this azole. Materials and methods: Cultured PBMCs were exposed to FNZ (6, 12, 30, 60 and 120 µg/mL) and the toxicological profile was assessed by the following parameters: cytotoxic and nuclear division index (necrotic, apoptotic and viable cells), DNA damage (alkaline comet test), mutagenic potential (micronucleus test), cytokine modulation (IL-1, IL-6, IL-10, TNF-α, IFN-γ), and predictive toxicity (Osiris® and LAZAR® programs). Results: Our results demonstrated that FNZ induced cellular DNA damage and mutagenicity at concentrations above the plasma peak (>30 µg/mL) and 6 µg/mL, respectively, which was associated with increased TNF-α, and decrease IL-6 and IL-10 concentrations. These effects may be related to increased apoptosis and cytotoxic nuclear division index in the cultured PBMCs. In silico results indicated potential mutagenic, tumorigenic, irritant, and carcinogenic effects, which were partially confirmed by the above assays. Discussion and conclusions: Together, these findings suggest the need to rationalize the use of FNZ, especially if it is used for long periods or with concomitant pathologies requiring azole therapy that may increase FNZ's plasma concentration.

Methylmalonate Induces Inflammatory and Apoptotic Potential: A Link to Glial Activation and Neurological Dysfunction.

Methylmalonic acid (MMA) accumulates in tissues in methylmalonic acidemia, a heterogeneous group of inherited childhood diseases characterized by neurological dysfunction, oxidative stress and neuroinflammation; it is associated with degeneration of striatal neurons and cerebral cortical atrophy. It is presently unknown, however, whether transient exposure to MMA in the neonatal period is sufficient to trigger inflammatory and apoptotic processes that lead to brain structural damage. Here, newborn mice were given a single intracerebroventricular dose of MMA at 12 hours after birth. Maze testing of 21- and 40-day-old mice showed that MMA-injected animals exhibited deficit in the working memory test but not in the reference test. MMA-injected mice showed increased levels of the reactive oxygen species marker 2',7'-dichlorofluorescein diacetate, tumor necrosis factor, interleukin-1ß, caspases 1, 3, and 8, and increased acetylcholinesterase activity in the cortex, hippocampus and striatum. This was associated with increased astrocyte and microglial immunoreactivity in all brain regions. These findings suggest that transient exposure to MMA may alter the redox state and cause neuroinflammatory/apoptotic processes and glial activation during critical periods of brain development. Similar processes may underlie brain dysfunction and cognitive impairment in patients with methylmalonic acidemia.

Guanosine Exerts Neuroprotective Effect in an Experimental Model of Acute Ammonia Intoxication.

The nucleoside guanosine (GUO) increases glutamate uptake by astrocytes and acts as antioxidant, thereby providing neuroprotection against glutamatergic excitotoxicity, as we have recently demonstrated in an animal model of chronic hepatic encephalopathy. Here, we investigated the neuroprotective effect of GUO in an acute ammonia intoxication model. Adult male Wistar rats received an intraperitoneal (i.p.) injection of vehicle or GUO 60 mg/kg, followed 20 min later by an i.p. injection of vehicle or 550 mg/kg of ammonium acetate. Afterwards, animals were observed for 45 min, being evaluated as normal, coma (i.e., absence of corneal reflex), or death status. In a second cohort of rats, video-electroencephalogram (EEG) recordings were performed. In a third cohort of rats, the following were measured: (i) plasma levels of glucose, transaminases, and urea; (ii) cerebrospinal fluid (CSF) levels of ammonia, glutamine, glutamate, and alanine; (iii) glutamate uptake in brain slices; and (iv) brain redox status and glutamine synthetase activity in cerebral cortex. GUO drastically reduced the lethality rate and the duration of coma. Animals treated with GUO had improved EEG traces, decreased CSF levels of glutamate and alanine, lowered oxidative stress in the cerebral cortex, and increased glutamate uptake by astrocytes in brain slices compared with animals that received vehicle prior to ammonium acetate administration. This study provides new evidence on mechanisms of guanine-derived purines in their potential modulation of glutamatergic system, contributing to GUO neuroprotective effects in a rodent model of by acute ammonia intoxication.

High Intensity Interval Training Reduces the Levels of Serum Inflammatory Cytokine on Women with Metabolic Syndrome.

Objectives: This study investigate the effects of a high intensity interval training (HIIT) and 2 weeks of detraining in functional and body composition parameters, lipoproteins, glucose metabolismand inflammation markers in postmenopausal women with metabolic syndrome (MS). Design: 17 untrained women with MS underwent a HIIT program for 12 weeks. Methods: The training was performed in treadmills, 3 days per week, with intensity ranging from 70-90% of the maximum heart rate (HRmax) and 2 weeks untrained (inactive). Functional and body composition parameters were evaluated before and after the training, while maximal oxygen uptake, lipoprotein and inflammation markers were analyzed before, after training and also in detraining. Results: The HITT program resulted in changesparameters as glucose, HbA1cand NOx after training. In addition, a reduction in pro-inflammatory interleukins and an increase in IL-10 after the HIIT program were found. However, an increase in plasma levels of lipoprotein was found and body composition parameters remain unaltered.Besides, only 2 weeks of detraining are able to revert the effects on inflammatory parameters afforded by the HIIT program. Conclusions: The HIIT program used here positively affected inflammatory profile and other parameters, as glucose, HbA1cand NOx, on postmenopausal women with MS. Moreover, 2 weeks of detraining can reverse the beneficial effects of HIIT program. Our results point out the necessity to aply acontinuous HITT program, in order maintain the benefits detected, to post menopausal women with MS.

The effects of rosuvastatin on lipid-lowering, inflammatory, antioxidant and fibrinolytics blood biomarkers are influenced by Val16Ala superoxide dismutase manganese-dependent gene polymorphism.

Rosuvastatin is a cholesterol-lowering drug that also attenuates the inflammatory process and oxidative stress via the reduction of superoxide anion production. Superoxide anions are metabolized by manganese-dependent superoxide dismutase (MnSOD or SOD2) in the mitochondria. In humans, there is a gene polymorphism where a change of alanine (Ala) to valine (Val) occurs at the 16th amino acid (Ala16Val-SOD2). The VV genotype has been associated with the risk of developing several metabolic diseases, such as hypercholesterolemia. Thus, to further explore this phenomenon, this study investigated the influence of the Val16Ala-SOD2 polymorphism on the lipid profile and inflammatory and fibrinolytic biomarkers of 122 hypercholesterolemic patients undergoing the first pharmacological cholesterol-lowering therapy who were treated with 20 mg rosuvastatin for 120 days. The findings indicate that the VV patients who present a low-efficiency SOD2 enzyme exhibit an attenuated response to rosuvastatin compared with the A-allele patients. The effect of rosuvastatin on inflammatory and fibrinolytic biomarkers was also less intense in the VV patients. These results suggest some pharmacogenetic effects of Val16Ala-SOD2 in hypercholesterolemia treatment.

Effects of Swimming on the Inflammatory and Redox Response in a Model of Allergic Asthma.

In this study we hypothesized that swimming during sensitization phase could result in a preventive effect in mice with allergic asthma. Swiss mice were divided into 4 groups: Control and Swimming (non-sensitized), OVA and OVA+Swimming (sensitized). The allergic inflammation was induced by 2 intraperitoneal injections and 4 aerosol challenges using ovalbumin. Swimming sessions were performed at high intensity over 3 weeks. 48 h after the last challenge mice were euthanized. Swimming decreased OVA-increased total IgE, IL-1, IL-4, IL-5 and IL-6 levels, as well as the number of total cells, lymphocytes and eosinophils in bronchoalveolar lavage fluid, (p<0.05). Simultaneously, swimming also increased IL-10 and glutathione levels in the Swimming and OVA+Swimming groups (p<0.05). The levels of glutathione peroxidase and catalase were increased only in the Swimming group when compared to all groups (p<0.05). 21 days of swimming resulted in an attenuation of pulmonary allergic inflammation followed by an increase of glutathione levels in the OVA group. Swimming only increased the levels of glutathione peroxidase and catalase in non-sensitized mice (p<0.05). These data suggest that the pulmonary anti-inflammatory effects produced by 3 weeks of high-intensity swimming in this model of OVA-induced asthma may be, at least partly, modulated by reduced oxidative stress and increased IL-10 production.

Association between advanced oxidation protein products and 5-year mortality risk among amazon riparian elderly population.

Proteins are important targets of several modifications caused by oxidative stress, leading to structural changes and consequently partial or total loss of their functions. The oxidized proteins include advanced oxidation protein products (AOPP) derived from oxidation-modified albumin, as well as fibrinogen and lipoproteins. An increase in AOPP levels indicates an oxidative stress state and the presence of coexisting inflammation. Several investigations have also suggested an association between high AOPP levels and aging-related diseases. However, the link between elevated AOPP levels and elderly mortality risk has not yet been investigated. Here, we report on a 5-year longitudinal study that investigated the potential association between AOPP levels and mortality using a population-based representative sample of riparian elders living in Brazilian Amazon region (Maués-AM). Age, sex, socioeconomic and cultural conditions, chronic morbidities, polypharmacy, and previous morbidities were also tested as potential confounders. The AOPP levels were measured in 540 (84.78%) individuals, all of whom were followed over a 5-year period in order to establish the mortality rate. Within this study period, 74 (13.7%) elders died and 466 (86.3%) survived. The AOPP levels were higher among the elders who died within the 5-year period (46.27 ± 40.6 mmol/L) compared with those who survived (36.79 ± 20.84 mmol/L) (p = 0.002). The analysis confirmed the link between high AOPP levels and mortality risk, independent of other intervenient factors. These results suggest that elevated AOPP levels could be used to predict mortality risk in elderly patients.

Estrogen plus progestin increase superoxide dismutase and total antioxidant capacity in postmenopausal women.

OBJECTIVE: This cross-sectional study aimed to evaluate the behavior of blood antioxidant enzymes (superoxide dismutase (SOD), catalase and glutathione peroxidase), plasma total antioxidant capacity and oxidative damage (lipid oxidation and protein carbonyl levels) and their relationship with the serum levels of steroid hormones in premenopausal and postmenopausal women without and with estrogen alone (ET) or estrogen plus progestin therapy (EPT). METHODS: Blood was collected from four groups of subjects: premenopausal women (n = 24), postmenopausal women without hormone therapy (n = 31), postmenopausal women with ET (n = 12) and postmenopausal women with EPT (n = 16). RESULTS: The activities of the different SOD isoforms (CuZnSOD and MnSOD) and the plasma total antioxidant power were significantly higher in the postmenopausal women under EPT than in the postmenopausal women without hormone replacement therapy (HRT). Only CuZnSOD activity was increased in women receiving ET compared to the postmenopausal women without HRT. However, no differences were observed in the levels of lipid or protein oxidation or in the non-enzymatic plasma antioxidants (uric acid and albumin) among the groups. The duration of HRT and serum estrogen levels were positively correlated to the blood CuZnSOD activity and to plasma total antioxidant power, whereas the serum progesterone levels were positively correlated to CuZnSOD activity and negatively correlated to protein carbonyl groups. Interestingly, the total antioxidant power of plasma was positively correlated to CuZnSOD and glutathione peroxidase activities. CONCLUSION: We conclude that EPT increases blood MnSOD and CuZnSOD activity in postmenopausal women, leading to an increased plasma total antioxidant capacity. This finding may be relevant to the prevention of oxidative stress-related disorders in postmenopausal women.

The effect of palm oil addition to the diet of dairy sheep on the immune response.

The aim of this study was to evaluate whether a diet based on palm oil has any influence on the immune response and on the number of eggs per gram of faeces (EPG) of gastrointestinal nematodes (GIN) in dairy sheep. To address this issue, 30 ewes in early lactation were confined and divided into three groups (n = 10) receiving a daily isoproteic and isoenergetic diet. Palm oil was added to the feed at different concentrations: 0% (control; group A), 4% (group B) and 6% (group C). The animals were treated with levamisole 10 days before the beginning of the experiment. Faecal samples were collected and analysed for EPG on day zero of the experiment. On days 60 and 120, individual faecal and blood samples were collected, and the FAMACHA(©) score for assessing clinical anaemia was carried out. The groups receiving palm oil showed a significant reduction in EPG in relation to the control group (A) on day 120. Serum immunoglobulin levels (IgG, IgM and IgE) and proinflammatory cytokine levels (TNF-α, IL-1 and IL-6) were significantly increased on days 60 and 120 (p < 0.05) in groups B and C. Therefore, these results suggest that palm oil stimulates the immune response in sheep, thus reducing EPG of GIN. The hypothesis that palm oil has direct anthelmintic activity should be tested in future studies.

In vitro effects of Ala16Val manganese superoxide dismutase gene polymorphism on human white blood cells exposed to methylmercury.

Environmental contamination by methylmercury (MeHg) is an enormous public health problem in world regions such as Amazonia. MeHg toxic effects seem to be influenced by environmental and genetic factors. However, few studies have evaluated the genetic influences of MeHg toxicity in humans. Therefore, the aim of this study was to evaluate the genetic influence of Ala16Val manganese superoxide dismutase gene polymorphism (Ala16Val-MnSOD) on the cytotoxic effects of in vitro human leukocytes exposed to MeHg. Subjects were selected from 100 individuals aged 26.4 ± 7.3 years genotyped to Ala16Val-MnSOD polymorphism (AA = 6, VV = 6, and AV = 12) to perform in vitro testing using white blood cells (WBCs). Reactive oxygen species production was measured using 2',7'-dichlorofluorescein diacetate fluorimetric assay, and cell viability was measured using MTT assay on WBC samples from the same subjects that were both exposed and not exposed to MeHg (2.5 µM for 6 h). The results showed that AA- and VV-WBCs exposed to MeHg did not display increased reactive oxygen species levels compared to those in cells that were not exposed. However, AV-leukocytes exposed to MeHg displayed increased ROS levels. Cellular viability comparison among genotypes exposed to MeHg showed that the viability of AA-WBCs was lower than that of VV-WBC, with mean values of 3.46 ± 0.13 and 3.08 ± 0.77 (standard error), respectively (P = 0.033), whereas heterozygous cells (AV) displayed intermediate values. This difference was likely due to the higher basal H2O2 production of AA-WBCs compared to that of other genotypes. These results suggest that the Ala16Val-MnSOD polymorphism has toxicogenetic effects in human cells exposed to MeHg.

Antioxidant and anti-inflammatory effects of quercetin in functional and morphological alterations in streptozotocin-induced diabetic rats.

The aim of this study was to investigate functional and morphological alterations caused by oxidative stress in streptozotocin (STZ)-induced diabetic rats and to evaluate the antioxidant effect of quercetin (QUE) in this disease. One hundred and thirty male Wistar rats, it were randomly distributed in 10 different experimental groups, with ten animals per group: Control Saline (CS), Control Ethanol (CE), Control QUE 5mg/kg (CQ5), Control QUE 25mg/kg (CQ25), Control QUE 50mg/kg (CQ50), Diabetic Saline (DS), Diabetic Ethanol (DE), Diabetic QUE 5mg/kg (DQ5), Diabetic QUE25 mg/kg (DQ25), Diabetic QUE 50mg/kg (DQ50). Therefore, hyperglycemia is directly involved in oxidative stress production, as well as in functional and morphological alterations caused by the excess of free radicals. QUE, specially at the dosage of 50mg/kg, can act as an antioxidant and anti-inflammatory agent, becoming a promising adjuvant in the treatment of diabetes mellitus.

Non-genomic, direct modulatory effect of 17ß-estradiol, progesterone and their synthetic derivatives on the activity of human erythrocyte CuZn superoxide dismutase.

This study aimed to evaluate whether natural or synthetic steroid hormones could directly modulate the activity of the different superoxide dismutase (SOD) isoforms found in human blood fractions without changing enzyme expression. Enzyme samples of human erythrocytes, the human platelet-rich plasma fraction (PRP) or isolated CuZnSOD, which was purified from human erythrocytes were pre-incubated with natural steroids (17ß-estradiol 17-acetate and progesterone) and their synthetic derivatives (ß-estradiol 3-benzoate and medroxyprogesterone 17-acetate). Then, CuZn and MnSOD activities were measured using the xanthine/xanthine oxidase/nitroblue tetrazolium method. Hormones had no effect on MnSOD activity from the PRP, but we show for the first time that natural and synthetic steroid hormones have a direct, bell-shaped effect on the activity of CuZnSOD from both male and female human erythrocytes. Low (physiological) hormone concentrations caused a dose-dependent increase in enzyme activity, which disappeared at higher hormone concentrations. In addition, the combination of synthetic and natural estrogens and progestins had a synergistic stimulatory effect on the activity of CuZnSOD from human erythrocytes. The molecular interaction between CuZnSOD and steroid hormones was preliminarily studied. Natural hormones did not change the electrophoretic mobility of SOD under denaturing conditions, but they did increase the absorption spectra of SOD in the 230-290 nm range. These data suggest that hormone-mediated modulation of CuZnSOD is related to subtle changes in protein conformation, possibly related to Trp and Phe residues. We propose that this effect may account for the physiological regulation of enzyme activity during conditions where steroid hormones undergo alterations as the ovulatory cycle.

Activity of ectonucleotidases and adenosine deaminase in rats exposed to cigarette smoke.

Cigarette smoke is a complex mixture of various toxic substances that are capable of initiating oxidative damage and promoting blood platelet alterations. In this study, we investigated the activities of the ectoenzymes NTPDase (ectonucleoside triphosphate diphosphohydrolase, CD39) and 5'-nucleotidase (CD73) in platelets as well as adenosine deaminase (ADA) in the plasma of rats exposed to aged and diluted sidestream smoke during 4 weeks. The rats were divided into two groups: I (control) and II (exposed to smoke). After the exposure period, blood was collected and the platelets and plasma were separated for enzymatic assay. The results demonstrated that NTPDase (with ATP as substrate) and 5'-nucleotidase (AMP as substrate) activities were significantly higher in group II (p < 0.05) as compared to group I, while no significant difference was observed for NTPDase with ADP as substrate. The ADA activity was significantly reduced in group II (p < 0.05) as compared with group I. Platelet aggregation was significantly increased in group II (p < 0.05) as compared with group I. We suggest that these alterations in the activity of enzymes from the purinergic system are associated with an increase in platelet aggregation. However, our study has demonstrated that the organism tries to compensate for this enhanced aggregation by increasing hydrolysis of AMP and reducing hydrolysis of adenosine, a potent inhibitor of aggregation and an important modulator of vascular tone.

Toxicity of South American snake venoms measured by an in vitro cell culture assay.

Cytotoxicity of venoms from eight medically important South American Crotalidae snakes (Bothrops and Lachesis genera) was determined, based on a procedure originally described for the screening of cytotoxic agents in general. The assay, the conditions of which were adapted to snake venoms, determines the survival of viable cells in monolayer culture upon exposure to the toxic agent. Snake venom toxicity was expressed as the venom dose that killed 50% of the cells (CT(50)) under the assay conditions. Bothrops neuwieddi mattogrossensis (CT(50)=4.74+/-0.35 microg/ml) and Bothrops leucurus (CT(50)=4.95+/-0.51 microg/ml) were the most cytotoxic whereas Bothrops atrox (CT(50)=34.64+/-2.38 microg/ml) and Bothrops sp. (CT(50)=33.89+/-3.89 microg/ml) were the least cytotoxic venoms, respectively. The relationship between CT(50) and other biological activities of these snake venoms was evaluated.

Primary culture of venom gland cells from the South American rattlesnake (Crotalus durissus terrificus).

Primary cultures of venom gland cells from the South American rattlesnake (Crotalus durissus terrificus) were attempted. At first, six different cell types were obtained including potentially secreting epithelial-like cells. Nonepithelial cell cultures were later invaded by fibroblast-like cells. Cultures of epithelial-like gland cells were successfully maintained, after testing different culture conditions by varying the media, incubation temperature, use of dissociating agents and adhesion substrates. The best results were achieved using plates precoated with rattlesnake skin collagen and incubation in CMRL 1415 modified for snake gland cells plus 10% fetal calf serum at 30 degrees C. The presence of venom could be demonstrated in the supernatant of five out of six epithelial-like gland cell cultures tested by ELISA, in the very first passages. After the third passage, however, venom amounts dropped to undetectable values. A total of 23 venom gland cell lines were obtained and are kept frozen in the laboratory; among them, five epithelial-like gland cell lines with up to 12 passages, that were continuously cultured for more than 30 weeks. The methodology described here was successfully applied to C. d. terrificus kidney cells culturing, developed to be used as negative control.

[Complete atrioventricular block in Kearns-Sayre syndrome]. / Bloqueio atrioventricular total em síndrome de Kearns-Sayre.

A thirty-three year old woman, known to have Kearns-Sayre syndrome for eight years, had an ECG pattern of right bundle branch block and left anterior fascicular block that evolved to complete atrioventricular block, leading her to a syncopal episode. A temporary pacemaker and a permanent one were installed. The patient has been asymptomatic so far.