Neurotoxicity is among the most harmful effects of xenobiotics. In this context, mercury (Hg) compounds (including methylmercury - MeHg) have triggered major concerns namely in terms of public health. Though Hg is recognised as a pernicious, persistent and ubiquitous contaminant in natural waters, the assessment of its potential to induce neuronal dysfunctions in aquatic animals environmentally exposed is an almost unexplored issue. Hence, the leitmotiv for the present project was the recognition of a knowledge gap concerning Hg neurotoxicity in fish, namely on identifying the underlying mechanisms and repercussions on brain function and individual behavior. Fish relevance in ecosystems and their high susceptibility to Hg exposure also point out the pertinence of this study. In fact, high levels of Hg were previously recorded in fish brain, which was elected as the organ that best reflected the environmental levels [1]. The well documented neurotoxicity to humans and rodents [2], allied to the similar results found in the few available studies on fish [3;4], pointed to the need to make scientific efforts on this direction.

Though Hg has been implicated in neurodegeneration [5], the mechanisms underlying its toxicity are still unclear, mainly in fish. In this context, it is particularly relevant to investigate the involvement of oxidative stress phenomena, since the Hg neurotoxicity was associated in mammalian systems and fish with the excessive generation of reactive oxygen species (ROS) and lipid peroxidation [6]. In fact, when compared to other organs, brain is especially endangered due to the high potential to generate ROS [7].

Therefore, the main research questions of the project are to: (1) clarify the Hg species (inorganic versus MeHg) mainly accumulated in the brain and the contribution of different uptake routes (water versus diet); (2) assess the role of oxidative stress in the Hg neurotoxicity; (3) search brain morphofunctional changes; (4) clarify if environmentally relevant levels of Hg (inorganic and MeHg) could induce behavior changes in fish. It will be investigated the causal relationships between point 1 and points 2, 3, 4, as well as the mechanistic association among points 2, 3 and 4. In addition, (5) the reversibility of the previous effects in a post-exposure period will be assessed in parallel with the progress in the Hg accumulated levels.

Keeping in view these central questions, an integrated approach was designed, involving laboratory experiments with exposure and post-exposure periods.  Exposures via water (inorganic Hg) and via food (inorganic and MeHg) will be performed either separately or in combination. Pursuing a more straightforward strategy to improve the knowledge on Hg toxicokinetics and toxicodynamics in fish, and particularly to clarify how inorganic Hg can be taken up in the central nervous system, the waterborne metal will be isotopically marked (as 201HgCl). The levels of inorganic Hg, 201Hg and MeHg in blood and accumulated in specific areas of the brain and spinal cord will be determined.

The influence of temperature (miming contrasting seasons, viz. winter and summer) in the uptake and fate of Hg species in fish body as well as on the brain biochemical and morphofunctional alterations will be considered.

Acetylcholinesterase will be determined in the brain, as well as a battery of oxidative stress endpoints (enzymatic and non-enzymatic antioxidants, total antioxidant capacity and damage of lipids and proteins). Additionally, histopathological and histochemical endpoints and the expression of several neurotransmitter receptors, as well as the evaluation of apoptosis and neurogenesis in the limbic system, will allow the assessment of morphofunctional alterations. Considering that behavioral impairments are integrated manifestations of biochemical, structural and functional disturbances, the locomotor and feeding activities of fish will be assessed.

The teleost Diplodus sargus (white seabream) will be adopted as test organism since it fulfils most requirements as bioindicator species.

The research team has vast expertise in the toxicity of various contaminants in fish including Hg [1;9;10], as well as on environmental chemistry of Hg [8] and neurosciences [11;12]. Furthermore, the team has a good knowledge of teleost as experimental model, supported by several years of investigations [e.g. 1;9;13]. The current project is an innovative research that could contribute to the elucidation of Hg neurotoxicity in fish and the proposed strategy is completely new in the context of ecotoxicology due to its integrative character, substantiated by the broad spectrum of repercussions addressed. Additionally, the application of sophisticated techniques of neurosciences is also an original aspect. Finally, the expected results will provide useful recommendations for policy-making, fundamental to (re)formulate regulatory processes for protecting the environment health.

Mário Pacheco
Principal Investigator

Patrícia A. Oliveira Pereira Kowalski
Researcher