Experimental Infection by Brucella ovis: Changes in NTPDase, 5'-Nucleotidase and Acetylcholinesterase Associated Cerebral Oxidative Stres

Géssica Perin, Anielen Dutra Silva, Nathieli Bianchi Bottari, Charles Elias Assmann, Teane Milagres Augusto Gomes, Mateus Fracasso, Matheus Dellaméa Baldissera, Aleksandro Schafer da Silva


Background: Changes in purinergic and cholinergic signaling have been demonstrated in various pathologies associated with inflammation; however, the changes in brucellosis caused by the Gram-negative coccobacillus Brucella ovis are not known. B. ovis is generally asymptomatic in sheep. Hepatosplenomegaly has been described in B. ovis, a non-zoonotic species, characterized by an extravascular inflammatory response. Purinergic system enzymes are closely involved with the modulation of the immune system, pro- and anti-inflammatory events. The objective of this study was to investigate the role of ectonucleotidases and cholinesterase’s in the brains of mice experimentally infected with B. ovis.

Materials, Methods & Results: Forty-eight animals were divided into two groups: control (n = 24) and infected (n = 24). In group infected, 100 µL containing 1.3 x 107 UFC B. ovis /mL via intraperitoneal was used in inoculation. The brains were collected from the animals on days 7, 15, 30 and 60 post-infection (PI). We measured levels of TBARS (substances reactive to thiobarbituric acid) and ROS (reactive oxygen species) in the brain. The activity of NTPDase (using ATP and ADP as substrate) and 5'-nucleotidase (using AMP as substrate) were evaluated in brain in addition to histopathological analysis. No histopathological lesions were observed in the control group nor the infected group at days 7, 15, and 30 PI. However,multifocal areas with moderate microgliosis and inflammatory infiltrates in the cerebral cortex were observed at day 60 PI in the infected animals. B. ovis DNA was detected in brain. During the course of infection, B. ovis caused greater lipid peroxidation in the brains of infected animals than in the control group at day 60PI. No significant results were observed at 7, 15 or day 30 PI. Similarly, there was significantly more reactive oxygen species at day 60 PI in brains of infected animals than in the control group. NTPDase activity (using ATP and AMP as substrate) was lower at days 7 and 15 PI in infected animals than in control. However, during the course of infection there was an increase in NTPDase activity at day 60 PI in the infected group. The infected animals showed a decrease of 5´-nucleotidase (AMP as substrate) activity at days 7 and 30 PI. On the other hand, 5´-nucleotidase activity was greater on day 60 PI in the experimental group than in the control. The results suggest that nucleotide hydrolysis was low in the acute phase (up to day 30 PI) due to the decrease of NTPDase and 5´-nucleotidase activities. After day 60 PI, there was a reversal in enzyme activities, probably with concomitant increase of extracellular nucleotides. AChE activity in brain on days 30 and 60 PI compared to control.

Discussion: Among the functions of NTPDase are inhibition of platelet aggregation, vascular homeostasis, modulation of inflammation and immune response, all via its regulation of extracellular concentrations of ATP, a pro-inflammatory molecule. E-NTPDase plays an important role in controlling lymphocyte function, including antigen recognition and activation of cytotoxic T cell effector functions, as well as the generation of signals. The enzyme E-5´-nucleotidase also exerts non-enzymatic functions, including induction of intracellular signaling and mediation of cell-cell adhesion and cell-matrix and migration. Levels of acetylcholine are regulated by cholinesterase enzymes that are present in cholinergic and noncholinergic tissues, as the acetylcholinesterase (AChE) is a membrane-bound enzyme, primarily found in the brain and cholinergic neurons, where it participates in the structural regulation of postsynaptic differentiation. The results demonstrated that the chronicity of infection by B. ovis causes oxidative damage and inflammation in the brain, as well as modulation of ectonucleotidases and AChE activities.

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Anglister L., Etlin A., Finkel E., Durrant A.R. & Lev-Tov A. 2008. Cholinesterases in development and disease. Chemico-Biological Interactions. 175(1): 92-100

Atkinson B., Dwyer K., Enjyoji K. & Robson S.C. 2006. Ecto-nucleotidases of the CD39/NTPDase family modulate platelet activation and thrombus formation: Potential as therapeutic targets. Blood Cells, Molecules & Diseases. 36(2): 217-222.

Bours M.J., Swennen E.L., Di Virgilio F., Cronstein B.N. & Dagnelie P.C. 2006. Adenosine 5’-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation. Pharmacology & Therapeutics. 112(4): 358-404.

Burnstock G. 2006. Pathophydiology and therapeutic potential of purinergic signaling. Pharmacology Review. 58(1): 58-86.

Chan K., Delfert D. & Junger K.D. 1986. A direct colorimetric assay for Ca2+- ATPase activity. Analytical Biochemistry. 157(3): 375-378.

Cornelius C., Crupi R., Calabrese V., Graziano A., Milone P. & Pennisi G. 2013. Traumatic brain injury: oxidative stress and neuroprotection. Antioxidants & Redox Signaling. 19(7): 836-853.

Chatonnet A. & Lockridge O. 1989. Comparision of butyrylcholinesterase and acetylcholinesterase. The Biochemical Journal. 260(5): 625-634.

Ellman G.L., Courtney D.K., Andres V. & Featherstone R.M. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology. 7(1): 88-95.

Gill D.A. 1931. Circling disease of sheep in New Zealand. Veterinary Journal. 87(1): 60-74.

Halliwell B. & Gutteridge M.C. 2007. Free Radicals in Biology and Medicine. 4th edn. Oxford: Oxford University Press, 896p.

Heymann D., Reddington M. & Kreutzberg G.M. 1984. Subcellular localization of 5’-nucleotidase in rat brain. Journal of Neurochemistry. 43(2): 263–273.

Hop H.T., Arayan L.T., Reyes A.W.B., Huy T.X.N., Min W.G. & Lee H.J. 2018. Heat-stress-modulated induction of NF-κB leads to brucellacidal pro-inflammatory defense against Brucella abortus infection in murine macrophages and in a mouse model. BMC Microbiology. 18(1): 44. doi: 10.1186/s12866-018-1185-9.

Jiang X., Leonard B., Benson R. & Baldwin C.L. 1993. Macrophage control of Brucella abortus: role of reactive oxygen intermediates and nitric oxide. Cellular Immunology. 151(4): 309-319.

Ohkawa H., Ohishi N. & Yagi K. 1978. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry. 95(4): 351-358.

Perin G., Jaguezeski A.M., Miglioranza D., da Silva T.M.A., Stefani L.M. & Frigo A. 2018. Hematological and biochemical disturbances caused by Brucella ovis infection using an experimental model. Comparative Clinical Pathology. 27(6): 1523-1530.

Rodriguez-Rodriguez A., Egea-Guerreiro J.J., Murillo-Cabezas F. & Carrillo-Vico A. 2014. Oxidative stress in traumatic brain injury. Current Medicinal Chemistry. 21(10): 1201-1211.

Rocha J.B.T., Emanuelli T. & Pereira ME. 1993. Effects of early undernutrition on kinetic parameters of brain acetylcholinesterase from adult rats. Acta Neurobiologiae Experimentalis. 53(3): 431-437.

Silva T.M.A., Paixão T.A., Costa É.A., Xavier M.N., Cortez Sá J. & Moustacas V.S. 2011. Putative ATP-Binding cassette transporter is essential for Brucella ovis pathogenesis in mice. Infection and Immunity. 79(4): 1706-1717.

Soreq H. & Seidman S. 2001. Acetylcholinesterase-new roles for and old actor. Nature Reviews Neuroscience. 2(2): 294-302

Schetinger M.R.C., Morsch V.M., Bonan C.D. & Wysec. T.S. 2007. NTPDase and 5'-nucleotidase activities under physiological and disease conditions: new perspectives for human health. BioFactors. 31(1): 77-98.

Schetinger M.R.C., Porto N.M., Moretto M.B., Morsch V.M., Rocha J.B.T. & Vieira V. 2000. New benzodiazepines alter acetylcholinesterase and ATPDase activities. Neuroscience Research. 25(7): 949-955.

Souza C.F., Baldissera M.D., Bottari N.B., Moreira K.L.S., da Rocha M.I.U.M., da Veiga M.L., Santos R.C.V. & Baldisserotto B. 2018. Purinergic signaling modulates the cerebral inflammatory response in experimentally infected fish with Streptococcus agalactiae: an attempt to improve the immune response. Molecular and Cellular Biochemistry. 443(2): 131-138.

DOI: https://doi.org/10.22456/1679-9216.91074

Copyright (c) 2019 Géssica Perin, Anielen Dutra Silva, Nathieli Bianchi Bottari, Charles Elias Assmann, Teane Milagres Augusto Gomes, Mateus Fracasso, Matheus Dellaméa Baldissera, Aleksandro Schafer da Silva

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