Exposure to Hg²⁺ and Pb²⁺ changes NTPDase and ecto-5′-nucleotidase activities in central nervous system of zebrafish (Danio rerio)

Abstract

Neurotransmission can be affected by exposure to heavy metals, such as mercury and lead. ATP is a signaling molecule that can be metabolized by a group of enzymes called ecto-nucleotidases. Here we investigated the effects of mercury chloride (HgCl₂) and lead acetate (Pb(CH₃COO)₂) on NTPDase (nucleoside triphosphate diphosphohydrolase) and ecto-5′-nucleotidase activities in zebrafish brain membranes. In vitro exposure to HgCl₂ decreased ATP and ADP hydrolysis in an uncompetitive mechanism and AMP hydrolysis in a non-competitive manner. Pb(CH₃COO)₂ inhibited ATP hydrolysis in an uncompetitive manner, but not ADP and AMP hydrolysis. In vivo exposure of zebrafish to HgCl₂ or Pb(CH₃COO)₂ (20 μg/L, during 24, 96 h and 30 days) caused differential effects on nucleotide hydrolysis. HgCl₂, during 96 h, inhibited the hydrolysis of ATP, ADP and AMP. After 30 days of exposure to HgCl₂, ATP hydrolysis returned to the control levels, ADP hydrolysis was strongly increased and AMP hydrolysis remained inhibited. Exposure to Pb(CH₃COO)₂ during 96 h caused a significant decrease only on ATP hydrolysis. After 30 days, Pb(CH₃COO)₂ promoted the inhibition of ATP, ADP and AMP hydrolysis. Semi-quantitative RT-PCR analysis showed no changes in the expression of NTPDase1 and 5′-nucleotidase, following 30 days of exposure to both metals. This study demonstrated that Hg²⁺ and Pb²⁺ affect the ecto-nucleotidase activities, an important enzymatic pathway for the control of purinergic signaling.

Introduction

Heavy metals, such as mercury and lead are important environmental contaminants, which can reach aquatic systems derived from effluents of industrial, urban and mining sources. These substances present severe risk to the aquatic biota and humans, even at sublethal concentrations (Baatrup, 1991, Jarup, 2003). Animals exposed to Hg²⁺ and Pb²⁺ have adverse developmental, reproductive, neurological and behavioral effects. Many cellular processes are affected by exposure to Hg²⁺ and Pb²⁺ and the correct function of central nervous system can be impaired by neurochemical changes (Aguilar and Kostrzewa, 2004). Synaptic transmission can be altered after exposure to these heavy metals. Changes in the release, extracellular metabolism and/or uptake and expression of components of neurotransmitter systems have been related to toxic effects observed in heavy metals-exposed animals (Cooper and Manalis, 1983).

ATP is a primitive signaling molecule that has been retained as a cotransmitter in every nerve type in both peripheral and central nervous system (Burnstock, 2004). This molecule is released to the synaptic cleft in a calcium-dependent manner, where it can act as a fast neurotransmitter or as a modulator, regulating the activity of other transmitter substances (Cunha and Ribeiro, 2000). ATP exerts its effects through purinoceptors, divided in two major classes, ionotropic P2X and metabotropic P2Y receptors (Ralevic and Burnstock, 1998). At the synapse, ATP can be metabolized by a group of enzymes called ecto-nucleotidases, which includes NTPDase family (nucleoside triphosphate diphosphohydrolase) and ecto-5′-nucleotidase. The final product of this enzyme cascade is the nucleoside adenosine, an important neuromodulator that acts on G-protein-coupled receptors, named A₁, A_2A, A_2B and A₃ (Ribeiro et al., 2003).

Ecto-nucleotidases are ubiquitous enzymes with a broad phylogenetic distribution, occurring in many vertebrate tissues. NTPDases present the ability to hydrolyze triphosphate and diphosphate nucleotides. Mammalian NTPDases1–3 and 8 are extracellular enzymes that can be classified according the ATP/ADP preference (Zimmermann, 2001, Bigonesse et al., 2004). NTPDase1 (CD39) hydrolyzes ATP and ADP almost equally well. NTPDase2 (CD39L1) has a large preference for ATP over ADP. NTPDase3 (CD39L3 or HB6) and NTPDase8 slightly prefer ATP over ADP by a ratio of about 3 and 2, respectively. The nucleotide AMP, which is the final product of ATP and ADP hydrolysis promoted by NTPDases, can be hydrolyzed by the action of an ecto-5′-nucleotidase, producing the neuromodulator adenosine. Ecto-5′-nucleotidase has a pivotal role together with the NTPDases in regulating the concentration of extracellular nucleotides and nucleosides to the purinoceptors (Zimmermann, 2001).

There are few studies demonstrating the effect of heavy metals on ecto-nucleotidases. Oliveira et al. (1994) investigated the in vitro and in vivo effect of HgCl₂ on synaptosomal ATP diphosphohydrolase from cerebral cortex of developing rats. These authors observed contrasting results, whereas ATP and ADP hydrolysis were inhibited in vitro, exposure in vivo did not affect the nucleotide hydrolysis. Furthermore, Moretto et al. (2004) verified the subchronic (0.1 mg/kg; 30 doses/30 days) effect of HgCl₂ on NTPDase and 5′-nucleotidase activity of adult rats, showing a significant increase on NTPDase activity, but not on 5′-nucleotidase activity.

Zebrafish is a consolidated model system in neuroscience and toxicological studies (Linney et al., 2004, Senger et al., 2005). The zebrafish genome project has demonstrated regions of syntenic relationship with human genome (Barbazuk et al., 2000). Purinoceptors were already identified in this teleost (Kucenas et al., 2003) and we characterized the presence of a NTPDase and an ecto-5′-nucleotidase activities in brain membranes of zebrafish (Rico et al., 2003, Senger et al., 2004). These enzymes were cation-dependent, with a maximal rate for nucleotide hydrolysis in a pH range of 7.5–8.0 in the presence of Ca²⁺ for NTPDase and Mg²⁺ for ecto-5′-nucleotidase (Rico et al., 2003, Senger et al., 2004).

Considering that mercury and lead are important environmental contaminants and previous studies have demonstrated the presence of purinergic receptors and enzyme activities involved in extracellular catabolism of nucleotides in zebrafish brain, the aim of present study was to investigate the effect of mercury chloride and lead acetate on NTPDase and ecto-5′-nucleotidase activities and expression in central nervous system of zebrafish.