Resistance Exercise Training Improves Metabolic and Inflammatory Control in Adipose and Muscle Tissues in Mice Fed a High-Fat Diet.

This study investigates whether ladder climbing (LC), as a model of resistance exercise, can reverse whole-body and skeletal muscle deleterious metabolic and inflammatory effects of high-fat (HF) diet-induced obesity in mice. To accomplish this, Swiss mice were fed for 17 weeks either standard chow (SC) or an HF diet and then randomly assigned to remain sedentary or to undergo 8 weeks of LC training with progressive increases in resistance weight. Prior to beginning the exercise intervention, HF-fed animals displayed a 47% increase in body weight (BW) and impaired ability to clear blood glucose during an insulin tolerance test (ITT) when compared to SC animals. However, 8 weeks of LC significantly reduced BW, adipocyte size, as well as glycemia under fasting and during the ITT in HF-fed rats. LC also increased the phosphorylation of AktSer473 and AMPKThr172 and reduced tumor necrosis factor-alpha (TNF-α) and interleukin 1 beta (IL1-ß) contents in the quadriceps muscles of HF-fed mice. Additionally, LC reduced the gene expression of inflammatory markers and attenuated HF-diet-induced NADPH oxidase subunit gp91phox in skeletal muscles. LC training was effective in reducing adiposity and the content of inflammatory mediators in skeletal muscle and improved whole-body glycemic control in mice fed an HF diet.

P2Y2 receptor activation promotes esophageal cancer cells proliferation via ERK1/2 pathway.

Esophageal cancer is a prominent worldwide illness that is divided into two main subtypes: esophageal squamous cell carcinoma and esophageal adenocarcinoma. Mortality rates are alarming, and the understanding of the mechanisms involved in esophageal cancer development, becomes essential. Purinergic signaling is related to many diseases and among these various types of tumors. Here we studied the effects of the P2Y2 receptor activation in different types of esophageal cancer. Esophageal tissue samples of healthy controls were used for P2Y2R expression quantification. Two human esophageal cancer cell lines Kyse-450 (squamous cell carcinoma) and OE-33 (adenocarcinoma) were used to perform in vitro analysis of cell proliferation, migration, adhesion, and the signaling pathways involved in P2Y2R activation. Data showed that P2Y2R was expressed in biopsies of patients with ESCC and adenocarcinoma, as well as in the two human esophageal cancer cell lines studied. The RT-qPCR analysis demonstrated that OE-33 cells have higher P2RY2 expression than Kyse-450 squamous cell line. Results showed that P2Y2R activation, induced by ATP or UTP, promoted esophageal cancer cells proliferation and colony formation. P2Y2R blockage with the selective antagonist, AR-C 118925XX, led to decreased proliferation, colony formation and adhesion. Treatments with ATP or UTP activated ERK 1/2 pathway in ESCC and ECA cells. The P2Y2R antagonism did not alter the migration of esophageal cancer cells. Interestingly, the esophageal cancer cell lines presented a distinct profile of nucleotide hydrolysis activity. The modulation of P2Y2 receptors may be a promising target for esophageal cancer treatment.

Adverse effects of p-TSA-doped polypyrrole particulate exposure during zebrafish (Danio rerio) development.

Nanomaterials have been attracting attention due to the wide range of applications in nanomedicine. Polypyrrole (PPy), a conductive polymer, has been employed in the biomedical field due to its stimulus-responsive properties, although in vivo studies to assess its potential undesirable effects are limited. This study evaluated the effects of PPy doped with p-toluene sulfonic acid ((p-TSA); PPy/p-TSA) exposure (at 25, 100, 250 and 500 µg/mL) during six consecutive days on mortality, hatching, spontaneous movement, heart rate, morphology and locomotion behavior of zebrafish embryos/larvae. Additionally, PPy/p-TSA envelopment of developing embryo chorions and gene expression of a hypoxia-related marker in this context were also evaluated. No significant mortality was found; however, altered heart rate and early hatching was identified in all exposed groups at 48 hours post-fertilization (hpf). Surprisingly, with the 500 µg/mL dose, hatching initiated as early as 24 hpf. PPy/p-TSA adhered to and enveloped the chorion of embryos in a time- and dose-dependent fashion; morphological changes in body length and ocular distance were found with higher concentrations. PPy/p-TSA-exposed animals showed locomotor behavioral alterations compatible with hypoactivity. A significant increase in the turn angle with a concomitant reduction in meander was also verified at higher concentrations. Taken together, these results emphasize the adverse effects of PPy/p-TSA on zebrafish development and behavior. Some effects of PPy/p-TSA exposure were dose-dependent, and indicate specific adverse effects of PPy/p-TSA on zebrafish development and behavior.

Antioxidants Reverse the Changes in the Cholinergic System Caused by L-Tyrosine Administration in Rats.

Tyrosinemia type II is an inborn error of metabolism caused by a deficiency in the activity of the enzyme tyrosine aminotransferase, leading to tyrosine accumulation in the body. Although the mechanisms involved are still poorly understood, several studies have showed that higher levels of tyrosine are related to oxidative stress and therefore may affect the cholinergic system. Thus, the aim of this study was to investigate the effects of chronic administration of L-tyrosine on choline acetyltransferase activity (ChAT) and acetylcholinesterase (AChE) in the brain of rats. Moreover, we also examined the effects of one antioxidant treatment (N-acetylcysteine (NAC) + deferoxamine (DFX)) on cholinergic system. Our results showed that the chronic administration of L-tyrosine decreases the ChAT activity in the cerebral cortex, while the AChE activity was increased in the hippocampus, striatum, and cerebral cortex. Moreover, we found that the antioxidant treatment was able to prevent the decrease in the ChAT activity in the cerebral cortex. However, the increase in AChE activity induced by L-tyrosine was partially prevented the in the hippocampus and striatum, but not in the cerebral cortex. Our results also showed no differences in the aversive and spatial memory after chronic administration of L-tyrosine. In conclusion, the results of this study demonstrated an increase in AChE activity in the hippocampus, striatum, and cerebral cortex and an increase of ChAT in the cerebral cortex, without cognitive impairment. Furthermore, the alterations in the cholinergic system were partially prevented by the co-administration of NAC and DFX. Thus, the restored central cholinergic system by antioxidant treatment further supports the view that oxidative stress may be involved in the pathophysiology of tyrosinemia type II.

Mechanisms underlying the antiproliferative effects of a series of quinoxaline-derived chalcones.

The present study aimed to characterize the effects of quinoxaline-derived chalcones, designed on the basis of the selective PI3Kγ inhibitor AS605240, in oral cancer cells. Three lead compounds, namely N9, N17 and N23, were selected from a series of 20 quinoxaline-derived chalcones, based on an initial screening using human and rat squamous cell carcinoma lineages, representing compounds with at least one methoxy radical at the A-ring. The selected chalcones, mainly N9 and N17, displayed marked antiproliferative effects, via apoptosis and autophagy induction, with an increase of sub-G1 population and Akt inhibition. The three chalcones displayed marked in vitro antitumor effects in different protocols with standard chemotherapy drugs, with acceptable toxicity on normal cells. There was no growth retrieval, after exposure to chalcone N9 alone, in a long-term assay to determine the cumulative population doubling (CPD) of human oral cancer cells. A PCR array evaluating 168 genes related to cancer and inflammation, demonstrated striking actions for N9, which altered the expression of 74 genes. Altogether, our results point out quinoxalinic chalcones, mainly N9, as potential strategies for oral cancer treatment.

Prevention of unpredictable chronic stress-related phenomena in zebrafish exposed to bromazepam, fluoxetine and nortriptyline.

RATIONALE: Several model organisms have been employed to study the impacts of stress on biological systems. Different models of unpredictable chronic stress (UCS) have been established in rodents; however, these protocols are expensive, long-lasting, and require a large physical structure. Our group has recently reported an UCS protocol in zebrafish with several advantages compared to rodent models. We observed that UCS induced behavioral, biochemical, and molecular changes similar to those observed in depressed patients, supporting the translational relevance of the protocol. OBJECTIVES: Considering that a pharmacological assessment is lacking in this zebrafish model, our aim was to evaluate the effects of anxiolytic (bromazepam) and antidepressant drugs (fluoxetine and nortriptyline) on behavioral (novel tank test), biochemical (whole-body cortisol), and molecular parameters (cox-2, tnf-α, il-6, and il-10 gene expression) in zebrafish subjected to UCS. RESULTS: We replicated previous data showing that UCS induces behavioral and neuroendocrine alterations in zebrafish, and we show for the first time that anxiolytic and antidepressant drugs are able to prevent such effects. Furthermore, we extended the molecular characterization of the model, revealing that UCS increases expression of the pro-inflammatory markers cox-2 and il-6, which was also prevented by the drugs tested. CONCLUSIONS: This study reinforces the use of zebrafish as a model organism to study the behavioral and physiological effects of stress. The UCS protocol may also serve as a screening tool for evaluating new drugs that can be used to treat psychiatric disorders with stress-related etiologies.

Omega-3 fatty acids are able to modulate the painful symptoms associated to cyclophosphamide-induced-hemorrhagic cystitis in mice.

This study investigated the effects of the long-term dietary fish oil supplementation or the acute administration of the omega-3 fatty acid docosahexaenoic acid (DHA) in the mouse hemorrhagic cystitis (HC) induced by the anticancer drug cyclophosphamide (CYP). HC was induced in mice by a single CYP injection (300mg/kg ip). Animals received four different diets containing 10% and 20% of corn or fish oil, during 21days. Separated groups received DHA by ip (1µmol/kg) or intrathecal (i.t.; 10µg/site) routes, 1h or 15min before CYP. The behavioral tests (spontaneous nociception and mechanical allodynia) were carried out from 1h to 6h following CYP injection. Bladder inflammatory changes, blood cell counts and serum cytokines were evaluated after euthanasia (at 6h). Immunohistochemistry analysis was performed for assessing spinal astrocyte and microglia activation or GPR40/FFAR1 expression. Either fish oil supplementation or DHA treatment (ip and i.t.) markedly prevented visceral pain, without affecting CYP-evoked bladder inflammatory changes. Moreover, systemic DHA significantly prevented the neutrophilia/lymphopenia caused by CYP, whereas this fatty acid did not significantly affect serum cytokines. DHA also modulated the spinal astrocyte activation and the GPR40/FFAR1 expression. The supplementation with fish oil enriched in omega-3 fatty acids or parenteral DHA might be interesting nutritional approaches for cancer patients under chemotherapy schemes with CYP.

GRPR/PI3Kγ: Partners in Central Transmission of Itch.

The gastrin-releasing peptide (GRP) and its receptor (GRPR) are important components of itch transmission. Upstream, but not downstream, aspects of GRPR signaling have been investigated extensively. We hypothesize that GRPR signals in part through the PI3Kγ/Akt pathway. We used pharmacological, electrophysiological, and behavioral approaches to further evaluate GRPR downstream signaling pathways. Our data show that GRP directly activates small-size capsaicin-sensitive DRG neurons, an effect that translates into transient calcium flux and membrane depolarization (∼ 20 mV). GRPR activation also induces Akt phosphorylation, a proxy for PI3Kγ activity, in ex vivo naive mouse spinal cords and in GRPR transiently expressing HEK293 cells. The intrathecal injection of GRP led to intense scratching, an effect largely reduced by either GRPR antagonists or PI3Kγ inhibitor. Scratching behavior was also induced by the intrathecal injection of an Akt activator. In a dry skin model of itch, we show that GRPR blockade or PI3Kγ inhibition reversed the scratching behavior. Altogether, these findings are highly suggestive that GRPR is expressed by the central terminals of DRG nociceptive afferents, which transmit itch via the PI3Kγ/Akt pathway. SIGNIFICANCE STATEMENT: Itch is the most common symptom of the skin and is related to noncutaneous diseases. It severely impairs patients' quality of life when it becomes chronic and there is no specific or effective available therapy, mainly because itch pathophysiology is not completely elucidated. Our findings indicate that the enzyme PI3Kγ is a key central mediator of itch transmission. Therefore, we suggest PI3Kγ as an attractive target for the development of new anti-pruritic drugs. With this study, we take a step forward in our understanding of the mechanisms underlying the central transmission of itch sensation.

High phosphate serum levels correlate with the severity of experimental severe acute pancreatitis: insight into the purinergic system.

OBJECTIVES: Extracellular purines are a component of the systemic inflammatory response, and their levels are modulated by ectonucleotidases. In addition, nucleotide hydrolysis releases phosphate. We studied serum phosphate levels as a predictor of severity in acute pancreatitis (AP) and their correlation with extracellular purinergic metabolism. METHODS: Acute pancreatitis was induced by the retrograde injection of sodium taurocholate. The AP group was compared with animals submitted to a model of sepsis by cecal ligation and puncture. The sham group was submitted to laparotomy and closure. We measured the phosphate and purine levels in serum and the expression of 5'-nucleotidase (CD73) and the adenosine A2a receptor in pancreatic tissue by quantitative real-time polymerase chain reaction. RESULTS: Serum phosphate levels were higher in severe AP and correlated with severity. Severe AP led to increased serum levels of adenosine diphosphate, adenosine monophosphate, and adenosine. In addition, adenosine monophosphate conversion to adenosine in serum was accelerated in the AP groups. We found a positive correlation between serum adenosine and phosphate in the AP groups. The expression levels of CD73 and the adenosine A2a receptor in the pancreas were not altered. CONCLUSIONS: Our study suggests that serum phosphate correlates with severity in AP and implicates extracellular purines in the systemic response to severe AP.

Biodegradation of [D-Leu1] microcystin-LR by a bacterium isolated from sediment of Patos Lagoon estuary, Brazil

Background: Toxic cyanobacterial blooms are recurrent in Patos Lagoon, in southern Brazil. Among cyanotoxins, [D-Leu1] microcystin-LR is the predominant variant whose natural cycle involves water and sediment compartments. This study aimed to identify and isolate from sediment a bacterial strain capable of growing on [D-Leu1] microcystin-LR. Sediment and water samples were collected at two distinct aquatic spots: close to the Oceanographic Museum (P1), in Rio Grande City, and on São Lourenço Beach (P2), in São Lourenço do Sul City, southern Brazil. Methods: [D-Leu1] microcystin-LR was isolated and purified from batch cultures of Microcystis aeruginosastrain RST9501. Samples of water and sediment from Rio Grande and São Lourenço do Sul were collected. Bacteria from the samples were allowed to grow in flasks containing solely [D-Leu1] microcystin-LR. This strain named DMSX was isolated on agar MSM with 8 g L−1 glucose and further purified on a cyanotoxin basis growth. Microcystin concentration was obtained by using the ELISA immunoassay for microcystins whereas bacterial count was performed by epifluorescence microscopy. The genus Pseudomonas was identified by DNA techniques. Results; Although several bacterial strains were isolated from the samples, only one, DMXS, was capable of growing on [D-Leu1] microcystin-LR. The phylogenetic analysis of the 16S rRNA gene from DMXS strain classified the organism as Pseudomonas aeruginosa. DMXS strain incubated with [D-Leu1] microcystin-LR lowered the amount of toxin from 1 μg.L−1 to < 0.05 μg.L−1. Besides, an increase in the bacterial count–from 71 × 105 bacteria.mL−1 to 117 × 105 bacteria.mL−1–was observed along the incubation. Conclusions: The use of bacteria isolated from sediment for technological applications to remove toxic compounds is viable. Studies have shown that sediment plays an important role as ...

Biodegradation of [D-Leu1] microcystin-LR by a bacterium isolated from sediment of Patos Lagoon estuary, Brazil

Background Toxic cyanobacterial blooms are recurrent in Patos Lagoon, in southern Brazil. Among cyanotoxins, [D-Leu1] microcystin-LR is the predominant variant whose natural cycle involves water and sediment compartments. This study aimed to identify and isolate from sediment a bacterial strain capable of growing on [D-Leu1] microcystin-LR. Sediment and water samples were collected at two distinct aquatic spots: close to the Oceanographic Museum (P1), in Rio Grande City, and on São Lourenço Beach (P2), in São Lourenço do Sul City, southern Brazil. Methods [D-Leu1] microcystin-LR was isolated and purified from batch cultures of Microcystis aeruginosastrain RST9501. Samples of water and sediment from Rio Grande and São Lourenço do Sul were collected. Bacteria from the samples were allowed to grow in flasks containing solely [D-Leu1] microcystin-LR. This strain named DMSX was isolated on agar MSM with 8 g L1 glucose and further purified on a cyanotoxin basis growth. Microcystin concentration was obtained by using the ELISA immunoassay for microcystins whereas bacterial count was performed by epifluorescence microscopy. The genus Pseudomonas was identified by DNA techniques. Results Although several bacterial strains were isolated from the samples, only one, DMXS, was capable of growing on [D-Leu1] microcystin-LR. The phylogenetic analysis of the 16S rRNA gene from DMXS strain classified the organism as Pseudomonas aeruginosa. DMXS strain incubated with [D-Leu1] microcystin-LR lowered the amount of toxin from 1 g.L1 to 0.05 g.L1. Besides, an increase in the bacterial countfrom 71×105 bacteria.mL1 to 117×105 bacteria.mL1was observed along the incubation. Conclusions The use of bacteria isolated from sediment for technological applications to remove toxic compounds is viable. Studies have shown that sediment plays an important role as a source of bacteria capable of degrading cyanobacterial toxins. This is the first Brazilian report on a bacteriumof the genus Pseudomonasthat can degrade [D-Leu1] microcystin-LR, the most frequent microcystin variant in Brazilian freshwaters.

Pharmacological inhibition of CXCR2 chemokine receptors modulates paraquat-induced intoxication in rats.

Paraquat (PQ) is an agrochemical agent commonly used worldwide, which is allied to potential risks of intoxication. This herbicide induces the formation of reactive oxygen species (ROS) that ends up compromising various organs, particularly the lungs and the brain. This study evaluated the deleterious effects of paraquat on the central nervous system (CNS) and peripherally, with special attempts to assess the putative protective effects of the selective CXCR2 receptor antagonist SB225002 on these parameters. PQ-toxicity was induced in male Wistar rats, in a total dose of 50 mg/kg, and control animals received saline solution at the same schedule of administration. Separate groups of animals were treated with the selective CXCR2 antagonist SB225002 (1 or 3 mg/kg), administered 30 min before each paraquat injection. The major changes found in paraquat-treated animals were: decreased body weight and hypothermia, nociception behavior, impairment of locomotor and gait capabilities, enhanced TNF-α and IL-1ß expression in the striatum, and cell migration to the lungs and blood. Some of these parameters were reversed when the antagonist SB225002 was administered, including recovery of physiological parameters, decreased nociception, improvement of gait abnormalities, modulation of striatal TNF-α and IL-1ß expression, and decrease of neutrophil migration to the lungs and blood. Taken together, our results demonstrate that damage to the central and peripheral systems elicited by paraquat can be prevented by the pharmacological inhibition of CXCR2 chemokine receptors. The experimental evidence presented herein extends the comprehension on the toxicodynamic aspects of paraquat, and opens new avenues to treat intoxication induced by this herbicide.

Mild hyperhomocysteinemia increases brain acetylcholinesterase and proinflammatory cytokine levels in different tissues.

Mild hyperhomocysteinemia is considered to be a risk factor for cerebral and cardiovascular disorders and can be modeled in experimental rats. Inflammation has been implicated in the toxic effects of homocysteine. Cholinergic signaling controls cytokine production and inflammation through the "cholinergic anti-inflammatory pathway," and brain acetylcholinesterase activity plays a role in this regulation. The aim of this present study is to investigate the effect of mild chronic hyperhomocysteinemia on proinflammatory cytokine levels in the brain, heart, and serum of rats. Activity, immunocontent, and gene expression of acetylcholinesterase in the brain and butyrylcholinesterase activity in serum were also evaluated. Mild hyperhomocysteinemia was induced in Wistar rats by homocysteine administration (0.03 µmol/g of body weight) twice a day, from the 30th to the 60th days of life. Controls received saline in the same volumes. Results demonstrated an increase in tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and the chemokine monocyte chemotactic protein-1 (MCP-1) in the hippocampus, as well as an increase in IL-1ß and IL-6 levels in cerebral cortex. Acetylcholinesterase activity was increased in rats subjected to mild hyperhomocysteinemia in both cerebral structures tested; the immunocontent of this enzyme was also increased in the cerebral cortex and decreased in the hippocampus. Levels of acetylcholinesterase mRNA transcripts were not altered. Peripherally, homocysteine increased TNF-α, IL-6, and MCP-1 levels in the heart and IL-6 levels in serum. Taken altogether, these findings suggest that homocysteine promotes an inflammatory status that can contribute, at least in part, to neuronal and cardiovascular dysfunctions observed in mild hyperhomocysteinemia.

Mild hyperhomocysteinemia reduces the activity and immunocontent, but does not alter the gene expression, of catalytic α subunits of cerebral Na+,K+-ATPase.

Na(+),K(+)-ATPase is a membrane protein which plays a key role in the maintenance of ion homeostasis that is necessary to neuronal excitability, secondary transport and neurotransmitter uptake. Mild hyperhomocysteinemia leads to several clinical manifestations and particularly cerebral diseases; however, little is known about the mechanisms of homocysteine on cerebral Na(+),K(+)-ATPase. In the present study, we investigated the effect of mild hyperhomocysteinemia on the activity, the immunocontent of catalytic subunits (α1, α2, and α3) and the gene expression of this enzyme. We used the experimental model of mild hyperhomocysteinemia that was induced by homocysteine administration (0.03 µmol/g of body weight) twice a day, from the 30th to the 60th postpartum day. Controls received saline in the same volumes. Results showed that mild hyperhomocysteinemia significantly decreased the activity and the immunocontent of the α 1 and α 2 subunits of the Na(+),K(+)-ATPase in cerebral cortex and hippocampus of adult rats. On the other hand, we did not observe any change in levels of Na(+),K(+)-ATPase mRNA transcripts in such cerebral structures of rats after chronic exposure to homocysteine. The present findings support that the homocysteine modulates the Na(+),K(+)-ATPase and this could be associated, at least in part, with the risk to the development of cerebral diseases in individuals with mild hyperhomocysteinemia.

Chronic administration of branched-chain amino acids impairs spatial memory and increases brain-derived neurotrophic factor in a rat model.

Maple syrup urine disease (MSUD) is a neurometabolic disorder that leads to the accumulation of branched-chain amino acids (BCAAs) and their α-keto branched-chain by-products. Because the neurotoxic mechanisms of MSUD are poorly understood, this study aimed to evaluate the effects of chronic administration of a BCAA pool (leucine, isoleucine and valine). This study examined the effects of BCAA administration on spatial memory and the levels of brain-derived neurotrophic factor (BNDF). We examined both pro-BDNF and bdnf mRNA expression levels after administration of BCAAs. Furthermore, this study examined whether antioxidant treatment prevented the alterations induced by BCAA administration. Our results demonstrated an increase in BDNF in the hippocampus and cerebral cortex, accompanied by memory impairment in spatial memory tasks. Additionally, chronic administration of BCAAs did not induce a detectable change in pro-BDNF levels. Treatment with N-acetylcysteine and deferoxamine prevented both the memory deficit and the increase in the BDNF levels induced by BCAA administration. In conclusion, these results suggest that when the brain is chronically exposed to high concentrations of BCAA (at millimolar concentrations) an increase in BDNF levels occurs. This increase in BDNF may be related to the impairment of spatial memory. In addition, we demonstrated that antioxidant treatment prevented the negative consequences related to BCAA administration, suggesting that oxidative stress might be involved in the pathophysiological mechanism(s) underlying the brain damage observed in MSUD.

L-tyrosine administration increases acetylcholinesterase activity in rats.

Tyrosinemia is a rare genetic disease caused by mutations on genes that codify enzymes responsible for tyrosine metabolism. Considering that tyrosinemics patients usually present symptoms associated with central nervous system alterations that ranges from slight decreases in intelligence to severe mental retardation, we decided to investigate whether acute and chronic administration of L-tyrosine in rats would affect acetylcholinesterase mRNA expression and enzymatic activity during their development. In our acute protocol, Wistar rats (10 and 30 days old) were killed one hour after a single intraperitoneal L-tyrosine injection (500 mg/kg) or saline. Chronic administration consisted of L-tyrosine (500 mg/kg) or saline injections 12 h apart for 24 days in Wistar rats (7 days old) and rats were killed 12 h after last injection. Acetylcholinesterase activity was measured by Ellman's method and acetylcholinesterase expression was carried out by a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay. We observed that acute (10 and 30 days old rats) and chronic L-tyrosine administration increased acetylcholinesterase activity in serum and all tested brain areas (hippocampus, striatum and cerebral cortex) when compared to control group. Moreover, there was a significant decrease in mRNA levels of acetylcholinesterase in hippocampus was observed after acute protocol (10 and 30 days old rats) and in striatum after chronic protocol. In case these alterations also occur in the brain of the patients, our results may explain, at least in part, the neurological sequelae associated with high plasma concentrations of tyrosine seen in patients affected by tyrosinemia type II.

Long-term proline exposure alters nucleotide catabolism and ectonucleotidase gene expression in zebrafish brain.

Hyperprolinemia is an inherited disorder of proline metabolism and hyperprolinemic patients can present neurological manifestations, such as seizures cognitive dysfunctions, and psychotic disorders. However, the underlying mechanisms of these symptoms are still unclear. Since adenine nucleotides play crucial roles in neurotransmission and neuromodulation, we evaluated the in vivo and in vitro effects of proline on ectonucleotidase activities and gene expression in zebrafish brain. For the in vivo studies, animals were exposed at two proline concentrations (1.5 and 3.0 mM) during 1 h or 7 days (short- or long-term treatments, respectively). For the in vitro assays, different proline concentrations (ranging from 3.0 to 1000 µM) were tested. Short-term proline exposure did not promote significant changes on the ectonucleotidase activities and gene expression. Long-term proline exposure significantly increased ATP catabolism in both concentrations tested (14 % and 22 %, respectively), whereas ADP and AMP hydrolysis were increased only at 3.0 mM proline (21 % and 17 %, respectively) when compared to control. Moreover, the relative gene expression of enpd3 increased in both treated groups after long-term proline, whereas enptd1 increased only at 3.0 mM proline. Proline in vitro did not promote significant changes on ectonucleotidase activities. Altogether, these data indicate that the enzymes responsible for the control of extracellular nucleotides levels might be altered after proline exposure in zebrafish, contributing to better understand the pathophysiology of this disease. Moreover, such findings might facilitate the use of the zebrafish as a complementary vertebrate model for studying inborn errors of amino acid metabolism.

Endotoxin-induced effects on nucleotide catabolism in mouse kidney.

Extracellular adenosine 5'-triphosphate (ATP) acts as a proinflammatory mediator. Adenosine, the final product of ATP breakdown, is an anti-inflammatory compound, acting mainly on adenosine A(2A) receptors. Considering that the kidney is an organ strongly affected during systemic inflammatory responses and that ectonucleotidases are responsible for the control of extracellular nucleotide and nucleoside levels, we examined the endotoxin-induced effects on ectonucleotidases in kidney membranes of mice, and whether CGS-21680 hydrochloride (3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoic acid), a selective adenosine A(2A) receptor agonist, antagonizes the lipopolysaccharide (LPS)-induced effects on nucleotide catabolism in kidney. Animals were injected intraperitoneally with 12 mg/kg LPS and/or 0.5mg/kg CGS-21680 or saline. Nucleotidase activities were determined in kidney membrane preparations and ATP metabolism was measured by high performance liquid chromatography (HPLC) assay. Analysis of ectonucleotidase expression was carried out by semi-quantitative semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Exposure to endotoxemia promoted an increase in ATP and p-Nitrophenyl thymidine 5'-monophosphate (p-Nph-5'-TMP) hydrolysis, and a decrease in adenosine 5'-monophosphate (AMP) hydrolysis. CGS-21680 treatment failed to reverse these changes. HPLC analysis indicated a decrease in extracellular ATP and adenosine levels in groups treated with LPS and LPS plus CGS-21680. The expression pattern of ectonucleotidases revealed an increase in Entpd3, Enpp2, and Enpp3 mRNA levels after LPS injection. These findings indicate that nucleotide and nucleoside availability in mouse kidney is altered at different stages of endotoxemia, in order to protect the integrity of this organ when exposed to systemic inflammation.

Chronic mild hyperhomocysteinemia alters ectonucleotidase activities and gene expression of ecto-5'-nucleotidase/CD73 in rat lymphocytes.

Since mild hyperhomocysteinemia is a risk factor for cardiovascular and cerebral diseases and extracellular nucleotides/nucleosides, which are controlled by the enzymatic action of ectonucleotidases, can induce an immune response, in the present study, we investigated the effect of chronic mild hyperhomocysteinemia on ectonucleotidase activities and expression in lymphocytes from mesenteric lymph nodes and serum of adult rats. For the chronic chemically induced mild hyperhomocysteinemia, Hcy (0.03 µmol/g of body weight) or saline (control) were administered subcutaneously from the 30th to the 60th day of life. Results showed that homocysteine significantly decreased ATP, ADP, and AMP hydrolysis in lymphocytes of adult rats. E-NTPDases transcriptions were not affected, while the ecto-5'-nucleotidase transcription was significantly decreased in mesenteric lymph nodes of hyperhomocysteinemic rats. ATP, ADP, and AMP hydrolysis were not affected by homocysteine in rat serum. Our findings suggest that Hcy in levels similar to considered risk factor to development of vascular diseases modulates the ectonucleotidases, which could lead to a pro-inflammatory status.

Acute restraint stress in zebrafish: behavioral parameters and purinergic signaling.

Despite the extensive knowledge about the effects of acute restraint stress (ARS) in rodents, zebrafish research is still elementary in this field, and the consequences of stress on purinergic system are unclear. Therefore, we evaluated the effects of ARS on behavior, biochemical, and molecular parameters in zebrafish brain. Animals were submitted to a 90 min ARS protocol and tested for anxiety levels, exploratory behavior, and memory performance. Furthermore, we analyzed ectonucleotidase and adenosine deaminase activities and their gene expression profile, as well as transcription of adenosine receptors. ARS increased anxiety, but did not impair locomotion or cognition. ARS significantly increased ATP hydrolysis, decreased cytosolic ADA activity, and changed the entpd and adora gene expression. In conclusion, ARS disturbed zebrafish behavior, and we hypothesize that the augmentation in adenosine-mediated signaling may be a strategy to reestablish homeostasis and normal behavior after a stressful event.