NTPDase and acetylcholinesterase activities in silver catfish , Rhamdia quelen ( Quoy & Gaimard , 1824 ) ( Heptapteridae ) exposed to interaction of oxygen and ammonia levels

The effects of various levels of oxygen saturation and ammonia concentration on NTPDase (ecto-nucleoside triphosphate diphosphohydrolase, E.C. 3.6.1.5) and acetylcholinesterase (AChE, E.C. 3.1.1.7) activities in whole brain of teleost fish (Rhamdia quelen) were investigated. The fish were exposed to one of two different dissolved oxygen levels, including high oxygen (6.5 mg.L-1) or low oxygen (3.5 mg.L-1), and one of two different ammonia levels, including high ammonia (0.1 mg.L-1) or low ammonia (0.03 mg.L-1) levels. The four experimental groups included the following (A) control, or high dissolved oxygen plus low NH3; (B) low dissolved oxygen plus low NH3; (C) high dissolved oxygen plus high NH3; (D) low dissolved oxygen plus high NH3. We found that enzyme activities were altered after 24 h exposure in groups C and D. ATP and ADP hydrolysis in whole brain of fish was enhanced in group D after 24 h exposure by 100% and 119%, respectively, compared to the control group. After 24 h exposure, AChE activity presented an increase of 34% and 39% in groups C and D, respectively, when compared to the control group. These results are consistent with the hypothesis that low oxygen levels increase ammonia toxicity. Moreover, the hypoxic events may increase blood flow by hypoxia increasing NTPDase activity, thus producing adenosine, a potent vasodilator.


Introduction
The biological response of an aquatic organism to pollutants frequently involves changes at cellular and biochemical levels, leading to changes in the structure and function of the cells, tissues, and behavior of the organism (Parvez & Raisuddin, 2005).Ammonia is a common aquatic pollutant and is toxic to fish (Miron et al., 2008).The main source of endogenous ammonia in fish is direct deamination of amino acids (Le-Ruyet et al., 1997).In fish, the endogenous ammonia concentration can also be increased by elevated concentrations of exogenous ammonia (Foss et al., 2007).In addition to un-ionized ammonia, other critical parameters determining water quality include dissolved oxygen, nitrite and carbon dioxide levels (Foss et al., 2007).While ammonia alone is highly toxic, ammonia that has interacted with low oxygen levels becomes more toxic (Wajsbrot et al., 1991;Foss et al., 2007).In fish, high ammonia levels cause hyperventilation (McKenzie et al., 1993), hyper-excitability, coma, convulsions and, finally, death (Wee et al., 2007).However, it has been suggested that oxygen levels above normal saturation might reduce ammonia toxicity (Colt et al., 1991).
The toxicity of ammonia to aquatic organisms has been attributed mainly to the un-ionized form (NH 3 ), while the ionized form (NH +  4 ) is considered less toxic (Thurston et al., 1984;Wood, 2001).In fact, biological membranes of freshwater teleosts are very permeable to un-ionized ammonia (NH 3 ) but not to NH + 4 (Alabaster & Lloyd, 1980).Moreover, it has been reported that the un-ionized form of ammonia freely crosses the blood-brain barrier by diffusion (Cooper et al., 1979).As a result, the increase in ammonia levels leads to severe central nervous system (CNS) dysfunctions in various organisms from fish to mammals (Viseck, 1968;Smart, 1976;Cooper & Plum, 1987;Butterworth, 1998;Atwood et al., 2000;Felipo & Butterworth, 2002).Many electrophysiological and biochemical mechanisms have been proposed to underlie the deleterious effects of ammonia on the CNS (Ronan et al., 2007).
In addition, ammonia plays a role in several biochemical processes and in enzymatic reactions, so alterations in ammonia levels can dramatically impact these processes.For example, increases in ammonia levels interfere with brain energy metabolism (Foss et al., 2007).The primary intracellular energy source is ATP, which also acts as an extracellular signaling molecule (Fields & Burnstock, 2006).Indeed, ATP is considered to be one of the most important neurotransmitters in the purinergic system (Fredholm, 1995).Purinergic signaling is responsible for modulating many of the signaling and biosynthetic processes in which extracellular nucleotides are involved, including vascular homeostasis, cell size maintenance, neuronal signaling, immune function, and protein and lipid modification (Brake & Julius, 1996;Burnstock, 1998;Gayle et al., 1998;Enjyoji et al., 1999;Marcus et al., 2003;Schweibert & Zsembery, 2003).Moreover, the NTPDase family, composed of the eight members NTPDase 1 to 8, is involved in controlling nucleotide and nucleoside concentrations, acting to regulate purinergic neurotransmission (Rico et al., 2008).Four members of the NTPDase family are typical cell surface-located enzymes with an extracellularly facing catalytic site (NTPDase1, 2, 3, 8).Thus, these nucleotidases constitute a highly refined system for regulating nucleotide-mediated signaling through ATP and/or ADP hydrolysis at the surface of many cells types (Robson et al., 2006;Rico et al., 2008).Additionally, following hydrolysis of ATP to AMP, AMP is dephosphorylated to adenosine by 5 'nucleotidase (Zimmermann, 1992).Adenosine is a good candidate for a metabolic signal because it is produced rapidly in response to decreasing oxygen and acts on specific receptors (A 1 , A 2A , A 2B and A 3 ) (Fredholm et al., 2001).Functionally, adenosine is a potent vasomodulator in mammalian and non-mammalian vertebrates, producing vasodilation in systemic, coronary and cerebral vessels (Lutz & Reiners, 1997;Mubagwa & Flameng, 2001).
ATP and the neurotransmitter acetylcholine (ACh) are coreleased and are inactivated enzymatically in the synaptic cleft by ectonucleotidases and cholinesterases, respectively (Gonçalves & Silva, 2007).ACh is critical to CNS function.Among the functions of ACh, it is involved in learning, memory, cortical organization of movement and control of cerebral blood flow (Schetinger et al., 1999).ATP acts as a cotransmitter and a modulator of cholinergic transmission, regulating ACh release by signaling through P2X and P2Y receptors (Cunha & Ribeiro, 2000;Dahm et al., 2006).The enzyme acetylcholinesterase (AChE, E.C. 3.1.1.7)is responsible for the hydrolysis of the ACh to choline and acetate in cholinergic synapses.In fish, AChE is enriched in brain and muscle tissues.Disturbances in AChE activity can affect locomotion and equilibrium in aquatic organisms and may impair feeding, escape, and reproductive behavior (Habig & Di Giulio, 1991;Saglio & Trijasse, 1998;Bretaud et al., 2000).The majority of information on brain and muscle AChE comes from inhibition of the enzyme, mainly through pesticide toxicity (Fernandez-Vega et al., 2002).When the enzyme is inhibited, ACh is not hydrolyzed in nerve synapses and neuromuscular junctions, causing build-up of ACh at these sites and leading to overstimulation of brain and muscular tissue (Roex et al., 2003).Because these previous studies were based on enzyme inhibition, the details of enzyme activation are little known in fish, although AChE activation likely affects ACh concentration in the synaptic cleft.Recently, reports have indicated that several pesticide classes, such as isooxazolidinone, quinoline, dichloropropionanilide and sulfonylurea enhance AChE activity (Miron et al., 2005;Moraes et al., 2007).
In this study, we studied silver catfish, Rhamdia quelen (Quoy & Gaimard, 1824, Siluriformes, Heptapteridae), because it is a native freshwater species of economical importance in southern Brazil due to its ability to survive cold winters and grow in the summer (Barcellos et al., 2004).We aimed to determine whether the interaction between two factors, oxygen saturation and ammonia levels, can alter the purinergic and cholinergic systems.To do so, we evaluated NTPDase and AChE activities in whole brain of silver catfish exposed to various oxygen and ammonia levels.

Animals and treatments
Juvenile silver catfish (156.1 ± 0.2 g, 28.2 ± 0.3 cm) obtained from a local fish farm were maintained in continuously aerated 250-L tanks for at least one week prior to experiments.Fish were maintained in darkness 24 h per day (except during feeding and cleaning of the tanks) as dark environments are favorable to silver catfish (Piaia et al., 1999).They were fed once a day with commercial feed for juvenile fish (Supra, 42% CP, Alisul Alimentos S.A., Carazinho, Brazil) until apparent satiety up to 24 h before the experiment.The methodology used in this experiment was approved by the Ethical and Animal Welfare Committee of the Universidade Federal de Santa Maria, protocol number: 073/2007.
Dissolved oxygen level and temperature (24.4 ± 0.1ºC) were monitored with an oxygen meter (model Y5512, YSI Inc., Yellow Springs, USA).Room temperature was maintained with an air conditioner.The pH level (7.38 ± 0.10) was verified with a DMPH-2 pH meter (Digimed, São Paulo, Brazil).Total ammonia level, including NH 3 and NH 4 + , was determined according to Boyd & Tucker (1992) and NH 3 level according to Piper et al. (1982).These parameters were measured every hour throughout the entire acclimation period.Dissolved oxygen levels were maintained through aeration with air and/or nitrogen, while NH 3 levels were reached by adding concentrated NH 4 Cl solution.Water hardness (19.33 ± 1.33 mg CaCO 3 L -1 ) was analyzed by the EDTA titrimetric method, and alkalinity (23.97 ± 0.6 mg CaCO 3 L -1 ) and nitrite level (maximum 0.07 mg L -1 ) was measured according to Boyd & Tucker (1992).These parameters were measured at the beginning and end of the experiment.
Fish were collected at 0, 6 and 24 h after exposure to the experimental conditions and dipped in ice-water slurry (2.4 kg ice : 3.6 L water) for 5 min to anaesthetize.The whole brain was excised, rapidly placed in a solution of 320 mM sucrose, 0.1 mM EDTA, 5 mM Tris-HCl, pH 7.5 (medium I) at 4ºC and weighed.Then, tissues were homogenized in 10 volumes of medium I in a motor-driven Teflon-glass homogenizer (15 strokes at 1000 rpm) and centrifuged for 10 min at 1000 x g.The supernatant was collected and used in the AChE assay.

Assay of NTPDase activity
From whole brain, NTPDase activity was determined using a reaction medium containing 5 mM KCl, 1.5 mM CaCl 2 , 0.1 mM EDTA, 10 mM glucose, 225 mM sucrose and 45 mM Tris-HCl buffer, pH 8.0, in a final volume of 200 µL, as described in a previous study from our laboratory (Schetinger et al., 2000).First, 20 µL of the enzyme preparation (8-10 µg of protein) was added to the reaction mixture and pre-incubated for 10 min at 37ºC.The reaction was initiated by the addition of ATP or ADP to a final concentration of 1.0 mM and proceeded for 20 min.The enzyme activity was expressed in nmol Pi/min.mg of protein.

Acetylcholinesterase assay for whole brain
AChE activity was assayed in the supernatant of the whole brain (S1).AChE activity was determined according to Ellman et al. (1961), modified by Rocha et al. (1993).The reaction mixture (2 mL final volume) was composed of 100 mM phosphate buffer, pH 7.5, and 1 mM 5,5'-dithio-bis-2nitrobenzoic acid (DTNB).The method is based on the formation of yellow anion, 4,4'-dithio-bis-2-nitrobenzoic, which was measured by absorbance at 412 nm during 2 min at 25ºC.The enzyme (40-50 µg of protein) was pre-incubated for 2 min, and the reaction was initiated by adding 0.8 mM acetylthiocholine iodide.Enzyme activity was expressed in µmoles AcSCh/h.mg of protein.

Protein determination
Protein was measured according to Bradford (1976) using bovine serum albumin as a standard.

Statistical analysis
To verify the homogeneity of variances, all data were submitted to a Levene test.As the variance of the data from each group were found to be equivalent, analysis was carried out using analysis of variance (three-way ANOVA) followed by the Tukey Kramer multiple comparisons test, and significance was set at a level of 95% (p < 0.05).All data were expressed as mean ± S.D.

Results
We found that NTPDase and AChE activities, as measured from the S1 fraction of whole brain, of silver catfish were altered only after 24 h of exposure to the experimental conditions.Specifically, we found a significant interaction between dissolved oxygen and ammonia levels on ATP and ADP hydrolysis as well as AChE activity (p < 0.05).
Statistical analysis using a Tukey-Kramer test revealed a significant increase (100%) in ATP hydrolysis in fish exposed to high NH 3 and low O 2 levels (group D) over the control group (A) after 24 h of exposure (Fig. 1).Additionally, post hoc analysis revealed that ADP hydrolysis increased 119% in group D compared to the control group after 24 h (Fig. 2).Two treatments in particular produced a significant increase in ADP hydrolysis in the whole brain, including high oxygen/ high ammonia and low oxygen/high ammonia, when compared to the control group.
AChE activity increased 34% in group D relative to the control group after 24 h exposure.In addition, fish exposed to high NH 3 and high O 2 levels (group C) showed a 39% increase in AChE activity over control after 24 h exposure (Fig. 3).
Based on the results obtained, 6 h exposure to low dissolved oxygen or high ammonia levels did not alter enzyme activities.

Discussion
In the present study, high exogenous un-ionized ammonia levels (NH 3 ) combined with various oxygen saturation levels altered NTPDase and AChE activities after 24 h of exposure.Several experiments have reported that ammonia interacts with oxygen, becoming highly toxic at low oxygen levels.At the cellular level, ammonia can interfere with energy metabolism through impairment of the tricarboxylic acid cycle in fish (Arillo et al., 1981), thus affecting brain energy metabolism.This outcome supports the hypothesis that ammonia is more toxic at low oxygen saturation levels.
The extracellular nucleotides ATP and ADP are involved in important physiological mechanisms, such as hemostasis and thromboregulation (Sarkis et al., 1986;Enjyoji et al., 1999) as well as neurotransmission (Edwards et al., 1992).The known ability of NTPDase to hydrolyze ATP and ADP nucleotides makes it a fundamental constituent in cellular metabolism (Schetinger et al., 2001).In this study, ATP and ADP hydrolysis were enhanced in the group exposed to low dissolved oxygen plus high NH 3 levels for 24 h.This finding supports the hypothesis that ammonia is more toxic at low oxygen saturation levels.
AChE activity was increased after 24 h exposure in both the high dissolved oxygen plus high NH 3 group C and the low dissolved oxygen plus high NH 3 group D compared to the control group.For the group that received low dissolved oxygen plus high NH 3 (group D), the change in NTPDase activity paralleled the change in AChE activity.This finding may be due to an interaction between low oxygen saturation and ammonia, resulting in intensified ammonia toxicity.Foss et al. (2007) determined that growth in juvenile turbots was not impaired when elevated ammonia levels were introduced in water supersaturated with oxygen, suggesting that hyperoxic conditions increase tolerance to an environment high in ammonia.This previous finding is supported by our finding of increased AChE activities in the high O 2 /high NH 3 fish compared with the low O 2 /high NH 3 fish (Fig. 3).
Importantly, ammonia can impair the tricarboxylic acid cycle in fish, which is responsible for acetyl-CoA oxidation to CO 2 , so increased NH 3 can lead to a build-up of acetyl-CoA.In this study, 24 h of exposure to low oxygen levels (hypoxia) plus high ammonia levels resulted in alterations of enzyme activities.Additionally, the increase in NTPDase activity was significantly greater that the increase in AChE activity.This differential increase in activities may be due to the increase of ammonia toxicity caused by an interaction with low oxygen saturation, as well as hypoxic events.It is known that hypoxic events lead to vasodilatation, which is promoted by adenosine, leading to increased blood flow and therefore substrate availability (Berne et al., 1983;Collis, 1989).
Our findings corroborate several studies reporting that high ammonia levels at normal or low oxygen saturation increase ammonia toxicity (Wee et al., 2007;Foss et al., 2007;Ronan et al., 2007).Specifically, our findings demonstrate that the interaction between low oxygen saturation and high   NH 3 levels results in increased NTPDase and AChE activities in whole brain of silver catfish after 24 h of exposure.

Fig. 1 .
Fig. 1.ATP hydrolysis in supernatant of whole brain of silver catfish.

Fig. 2 .
Fig. 2. ADP hydrolysis in supernatant of whole brain of silver catfish.

Fig. 3 .
Fig. 3. AChE activity in supernatant of whole brain of silver catfish.