Kinetic properties of E-NTPDase activity in lymphocytes isolated from bone marrow, thymus and mesenteric lymph nodes of Wistar rats.

Plasma membrane anchored nucleotidases (E-ATPDases), as the E-NTPDase family, could hydrolyze and regulate the pericellular levels of nucleotides in lymphocytes. Each immune organ has a different microenvironment and display lymphocytes with different functions and phenotypes. Considering the different functions of each resident subpopulations of lymphocytes, the E-ATPDases activities in bone marrow (BML), thymus (TL) and mesenteric lymph node (MLL) lymphocytes of Wistar rats were characterized. The hydrolysis of extracellular nucleotides (eATP and eADP) showed linearity in time of reaction between 0 and 120 min, and concentration of lymphocytes expressed in proteins between 1 and 6 µg protein in the reaction medium. The optimal activity was attained at 37 °C in a pH value of 8.0. The necessity of the cofactors Ca2+ and Mg2+ for the enzymatic activity was confirmed through a curve of concentration of 0-1000 µM in the reaction medium, with both cofactors showing similar effects in the enzymatic activity. The Chevillard plot revealed that the hydrolysis of eATP and eADP occurred at the same active site of the enzyme. The analyses of E-ATPDases inhibitor and enzyme specificity showed possible involvement of E-NTPDase isoforms - 1 and - 2 in the isolated cells. Furthermore, different kinetic behavior of the nucleotide hydrolysis in each resident subpopulation lymphocyte was observed in this study, as MLL showed the higher Vmax with the lowest km values, while TL had the lowest Vmax and high km values. The kinetic values for E-NTPDase activity of each immune tissue lymphocytes can be an important therapeutic target for numeral immune-related diseases.

Effects of clopidogrel bisulfate on B16-F10 cells and tumor development in a murine model of melanoma.

Metastatic melanoma is a very aggressive skin cancer. Platelets are constituents of the tumor microenvironment and, when activated, contribute to cancer progression, especially metastasis and inflammation. P2Y12 is an adenosine diphosphate receptor that triggers platelet activation. Inhibition of P2Y12 by clopidogrel bisulfate (CB) decreases platelet activation, which is also controlled by the extracellular concentration and the metabolism of purines by purinergic enzymes. We evaluated the effects of CB on the viability and proliferation of cultured B16-F10 cells. We also used a metastatic melanoma model with C57BL-6 mice to evaluate cancer development and purine metabolism modulation in platelets. B16-F10 cells were administered intraperitoneally to the mice. Two days later, the animals underwent a 12-day treatment with CB (30 mg/kg by gavage). We have found that CB reduced cell viability and proliferation in B16-F10 culture in 72 h at concentrations above 30 µm. In vivo, CB decreased tumor nodule counts and lactate dehydrogenase levels and increased platelet purine metabolism. Our results showed that CB has significant effects on melanoma progression.

Hydroalcoholic extract of leaf of Arachis hypogaea L. (Fabaceae) did not induce toxic effects in the repeated-dose toxicity study in rats.

Arachis hypogaea L. (peanut) leaf is traditionally used for the treatment of insomnia in Asia. However, studies describing the safety and toxicity profile for this plant preparation are limited. Thus, the goal of this study was to investigate the toxicity of peanut leaf hydroalcoholic extract (PLHE) repeated treatment. The extract was administered orally (100, 300 or 1000 mg/kg) in male and female Wistar rats for 28 days (OECD guideline 407). PLHE treatment did not cause mortality or weight variation in the animals. Also, there was no alteration on locomotor activity (open field test), motor coordination (rotarod test), or anxiety behaviour (elevated plus-maze test). Male rats had a reduction in relative liver weight (100 mg/kg) and an increase in total kidney weight (1000 mg/kg), but there was no change in biochemical and haematological parameters after PLHE treatment. Free extracellular double-stranded DNA (dsDNA) levels was also evaluated, but PLHE treatment did not increase this parameter in rat organs. Also, the dose of 1000 mg/kg of PLHE significantly increased the total thiols in the liver of females compared with the control animals. Thus, PLHE did not induce toxicity after repeated exposure for 28 days in rats.

Blood-brain barrier breakdown, memory impairment and neurotoxicity caused in mice submitted to orally treatment with thymol.

Several evidences have related the biochemical and pharmacological properties of thymol, but the possible neurotoxic effects of this compound remain unknown and not evaluated. Thus, the purpose of this study was to evaluate whether intake of thymol in different doses (10, 20 and 40 mg/kg) induce neurotoxicity and behavioral alterations using mice as experimental model, as well as the involvement of blood-brain barrier (BBB) and brain neurotransmitters in these alterations. Thymol (20 and 40 mg/kg) significantly decrease latency time to inhibitory avoidance task when compared to control group, indicating a memory loss after 30 days of oral treatment. Also, thymol (20 and 40 mg/kg) induced a significant increase on BBB permeability to Evan's blue dye when compared to control group, which is an indicative of BBB breakdown. Moreover, a significant increase of brain acetylcholinesterase (AChE) was observed in mice treated with 40 mg/kg of thymol, while the activity of sodium-potassium pump (Na+, K+-ATPase) was inhibited in mice treated with 20 and 40 mg/kg thymol when compared to control group. Finally, mice that received 20 and 40 mg/kg thymol showed a significant increase on cerebral reactive oxygen species (ROS) levels and cerebral xanthine oxidase (XO) activity compared to control group. Based on these evidences, the rupture of BBB can be considered an important pathway linked in thymol-induced memory loss. Also, the augmentation of brain ROS levels elicited by increase on XO activity may be a via involved in the damage to BBB, and an oxidative pathway that impairs the activity of brain neurotransmitters, as AChE and Na+, K+-ATPase. In summary, the dose of 10 mg/kg thymol can be safe and without neurotoxic effects in a period of 30 days of intake.

Ecto-enzymes activities in splenic lymphocytes of mice experimentally infected by Trypanosoma cruzi and treated with specific avian immunoglobulins: an attempt to improve the immune response.

The aim of this study was to evaluate the therapeutic efficacy of specific avian polyclonal antibodies (IgY) against Trypanosoma cruzi and their interaction with ecto-enzymes of the purinergic system (NTPDase and adenosine deaminase (ADA) activities) in splenic lymphocytes. For this, mice were divided into six groups: three non-infected (A, B, and C) and three infected (D, E, and F). The groups A and D were composed by negative and positive controls, respectively; while the groups B and E were treated prophylactically with IgY (50 mg/kg), and the groups C and F were treated therapeutically with IgY (50 mg/kg). Treatment with IgY reduced parasitemia on day 6 post-infection (PI) compared to the infected control group, but it was similar on day 8 PI. Moreover, infected and treated animals (the groups E and F) did not show neither amastigotes in the cardiac tissue nor cardiac lesions when compared to the positive control group (the group D). The E-NTPDase (ATP and ADP as substrate) and ADA activities in splenic lymphocytes increased significantly in the positive control group (the group D) compared to the negative control group (the group A). The therapeutic treatment of IgY (the group F) was able to prevent the increase of E-NTPDase and E-ADA activities compared to the positive control group (the group D), but this finding was not observed in animals that received the prophylactic treatment (the group E). The therapeutic treatment of IgY may be considered an interesting approach to improve the immune response of mice experimentally infected by T. cruzi.

Xanthine oxidase activity affects pro-oxidative and pro-inflammatory profiles in spleen of silver catfish experimentally infected with Aeromonas caviae.

Several pieces of evidence have linked the involvement of xanthine oxidase (XO), a source of uric acid and reactive oxygen species (ROS), to pro-oxidative and pro-inflammatory effects observed during bacterial fish diseases. Thus, the aim of this study was to evaluate whether upregulation of splenic XO activity contributes to disease pathogenesis of Aeromonas caviae infection, as well as whether it may be considered a pathway involved in ROS and nitric oxide (NO) production. XO activity increased in the spleen of infected animals, as did the splenic levels of uric acid, ROS and metabolites of nitric oxide (NOx), compared to the uninfected control group. Based on this evidence, upregulation of splenic XO activity induces pro-oxidant and pro-inflammatory profiles in the spleen of fish infected by A. caviae due to excessive formation of uric acid. Moreover, excessive uric acid induces the release of pro-inflammatory mediators, such as ROS and NOx, which contribute to disease pathophysiology. In summary, upregulation of XO activity may be considered a pathway involved in ROS and NOx production.

Peripheral blood mononuclear cells from rat model of pleurisy: The effects of hesperidin on ectoenzymes activity, apoptosis, cell cycle and reactive oxygen species production.

The present study investigates the effect of hesperidin; a flavonone commonly found in citrus fruits, on the ectoenzymes (ectonucleotidase and ecto-adenosine deaminase) activity, cell viability, apoptosis, cell cycle arrest and reactive oxygen species production in peripheral blood mononuclear cells (PBMCs) from rat model of pleurisy. Wistar rats were pretreated with either saline or hesperidin (80mg/kg) by oral gavage for 21days and injected intrapleurally with 2% carrageenan or saline on the 22nd day. PBMCs were subsequently prepared after 4h of carrageenan induction. The results revealed that hesperidin may exhibit its anti-inflammatory effects through possible modulation of ectonucleotidase (E-NTPDase) and ecto-adenosine deaminase (E-ADA) activities, reduction of intracellular reactive oxygen species, prevention of DNA damage and modulation of apoptosis as well as activation of cell cycle arrest. This study suggests some possible underlying anti-inflammatory mechanisms of hesperidin on PBMCs in acute inflammatory condition. Furthermore, hesperidin may minimize oxidative injury mediated pleurisy in rat.

Increased oxidative stress alters nucleosides metabolite levels in sickle cell anemia.

OBJECTIVES: This study was conducted to assess the markers of oxidative stress, myeloperoxidase (MPO), acetylcholinesterase (AChE) and xanthine oxidase (XO) activities as well as the levels of nucleotide metabolites in sickle cell anemia (SCA) patients. METHODS: Fifteen SCA treated patients and 30 health subjects (control group) were selected. The markers of oxidative stress (levels of reactive oxygen species (ROS), plasma proteins, carbonyl content, lipid peroxidation (TBARS), total thiols (T-SH), glutathione and catalase activity), MPO, AChE and XO activities as well as the levels of nucleotide metabolites were measured in SCA patients. RESULTS: ROS, thiobarbituric acid-reactive substances (TBARS) and T-SH levels as well as the activities of catalase and MPO were significantly increased while glutathione level was reduced in SCA patients. Furthermore, a significant (P < 0.001) increase in hypoxanthine level was demonstrated in SCA patients. However, the serum levels for xanthine (P < 0.01) and uric acid (P < 0.001) were decreased in SCA patients. A significant (P < 0.001) decrease in XO activity was detected in SCA patients. DISCUSSION: The altered parameters in SCA patients suggest that the generation and impairment of oxidative stress in this disease as well as antioxidant markers are contributory factors towards cellular redox homeostasis and alteration of purine metabolites.

Involvement of cholinergic and purinergic systems during the inflammatory response caused by Aeromonas hydrophila in Rhamdia quelen.

The aim of this study was to evaluate the cholinergic (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)) and purinergic (adenosine deaminase (ADA)) systems in head kidney, spleen, total blood and serum samples in experimentally infected fish with A. hydrophila, and the involvement of these systems during the inflammatory process. Silver catfish (Rhamdia quelen) juveniles were divided into two groups with seven fish each: uninfected (negative control) and infected (positive control). On day 2 post-infection, animals were euthanized and the head kidney, spleen, total blood and serum were collected. AChE and ADA activities in head kidney and spleen decreased in infected animals compared to uninfected animals, as well as AChE in total blood and seric ADA activities. BChE activity was not expressed in the evaluated tissues. Therefore, our results lead to the hypothesis that cholinergic and purinergic systems play an important role on the immune response against A. hydrophila with an anti-inflammatory effect. In summary, AChE and ADA activities reduced probably in order to protect against tissue inflammatory damage caused by infection.

Free and nanoencapsulated curcumin prevents cigarette smoke-induced cognitive impairment and redox imbalance.

Cigarette smoke-exposure promotes neurobiological changes associated with neurocognitive abnormalities. Curcumin, a natural polyphenol, have shown to be able to prevent cigarette smoke-induced cognitive impairment. Here, we investigated possible mechanisms involved in curcumin protection against cigarette smoke-induced cognitive impairment and, due to its poor bioavailability, we investigated the potential of using curcumin-loaded lipid-core nanocapsules (C-LNC) suspension. Rats were treated with curcumin and cigarette smoke, once a day, 5 days each week, for 30 days. Animals were divided into ten groups: I, control (vehicle/corn oil); II, curcumin 12.5mg/kg; III, curcumin 25mg/kg; IV, curcumin 50mg/kg; V, C-LNC 4 mg/kg; VI, tobacco exposed; VII, curcumin 12.5mg/kg along with tobacco exposure; VIII, curcumin 25mg/kg along with tobacco exposure; IX, curcumin 50mg/kg along with tobacco exposure; X, C-LNC 4 mg/kg along with tobacco exposure. Cigarette smoke-exposure impaired object recognition memory (P<0.001), indicated by the low recognition index, increased biomarkers of oxidative/nitrosative stress such as TBARS (P<0.05) and NOx (P<0.01), decreased antioxidant defenses such as NPSH content (P<0.01) and SOD activity (P<0.01) and inhibited the activities of enzymes involved in ion homeostasis such as Na(+),K(+)-ATPase and Ca(2+)-ATPase. Both curcumin formulations (free and nanoencapsulated) prevented the memory impairment, the redox imbalance and the alterations observed in the ATPases activities. Maintenance of ion homeostasis and redox balance is involved in the protective mechanism of curcumin against tobacco-induced cognitive impairment. Our results suggest that curcumin is a potential therapeutic agent for neurocognition and that C-LNC may be an alternative to its poor bioavailability.