Environmentally relevant manganese concentrations evoke anxiety phenotypes in adult zebrafish
Graphical Abstract
Introduction
Manganese (Mn) is one of the most abundant elemental metals in the earth's crust, existing as inorganic and organic forms, and also in several chemical and complexed states. The element is used for various industrial purposes, including iron and steel production, manufacture of batteries, glass, ceramics, adhesives, paints, matches, and fireworks (Aschner and Aschner, 2005). Besides, organic forms can be found in the fuel additive methyl-cyclopentadienyl manganese tricarbonyl (MMT), in fungicides (e.g., Maneb and Mancozeb), and in contrast agents for magnetic resonance imaging (Santamaria et al., 2007).
Mn is found in all mammalian tissues where it is required for the normal amino acid, lipid, protein, and carbohydrate metabolism. It is also essential for the antioxidant and immune responses of cells (Avila et al., 2013, Chen et al., 2019). Enzymes as arginase, agmatinase, glutamine synthetase, and Mn superoxide dismutase (MnSOD) require Mn as a co-factor (Balachandran et al., 2020, Bowman et al., 2011, Erikson and Aschner, 2019).
For humans, the adult dietary intake of Mn has been estimated to range from 0.9 to 10 mg Mn/day; being the recommended daily intake ± 2 mg/day. In potable water, the concentration of Mn may vary from 1 to 100 µg/L (Avila et al., 2013, Farina et al., 2013; Institute of Medicine (US), 2001). However, examples of drinking water or potential drinking-water supplies with Mn concentrations > 400 μg/L can be found worldwide (Frisbie et al., 2015). The levels of Mn considered safety for the aquatic communities vary from 0.1 to 0.5 mg/L; but higher levels have been found in the aquatic ecosystems due to the large inputs of Mn from human activities (Queiroz et al., 2021).
Despite its essentiality, the excess of Mn can be toxic to the organisms. Ingestion of foods and water containing high levels of Mn, and occupational exposure are the main sources of contamination for the people (Balachandran et al., 2020, Peres et al., 2016). Excessive intake and exposure to Mn have already been associated with neurological, reproductive, and respiratory disorders (Aschner et al., 2005, Bowler et al., 2007).
In the brain, Mn accumulates mainly in the basal ganglia region, inducing the neurodegenerative illness known as Manganism, a syndrome similar to Parkinson’s disease (PD), and characterized by psychiatric and motor impairments. Manganism symptoms include ataxia, dementia, and anxiety (Bowler et al., 2007, Gerber et al., 2002, Peres et al., 2016). Mounting evidence has suggested that Mn is also a risk factor for other neurodegenerative diseases, including Alzheimer’s disease, Amyotrophic lateral sclerosis, and Huntington’s disease (Avila et al., 2013, Bouabid et al., 2016, Sachse et al., 2019).
Considering the dual role of Mn, it is extremely important to carry out toxicological studies that investigate the limits between the functional and toxicological window of exposure to the element in the biological systems. In this scenario, the SNC has been the main target of investigations; however, the neurological networks and manifestations triggered by Mn toxicity are not still clear.
Zebrafish (Danio rerio) has emerged as a feasible organism model for translational in neuroscience research. In addition to conserved brain architecture, the fish exhibits a complex behavioral repertoire that mirrors those found in mammals (Fontana et al., 2018, Kalueff et al., 2013).
Then, using zebrafish, this study was aimed to verify whether in addition to locomotor deficits, Mn in environmentally relevant concentrations could cause psychiatric disturbances, namely anxiety-phenotypes. In order to find biochemical endpoints following Mn exposure, we measured AChE enzyme activity and cortisol levels of fish. We hypothesized that Mn induces neurobehavioral alterations and neurotoxicity in fish, events marked by cortisol and Ache activity dysregulation.
Section snippets
Animals
Adult wild-type zebrafish (Danio rerio) (4–6 months-old, males and females, approximately 50:50 ratio) were obtained from a local supplier (Hobby Aquarios, RS, Brazil). Fishes were maintained in 40 L aquariums (at a maximum density of 2 fish per liter) for at least 2 weeks before the experiments. The tanks contained non-chlorinated water under constant aeration and filtration at 25 ± 2 °C, pH = 7.0 – 8.0 were maintained on a 14/10 h photoperiod (light/dark cycle). Fish were fed twice daily with
Manganese alters the locomotor and exploratory activity of zebrafish in the novel tank test
The results from Fig. 1 illustrate the locomotor and exploratory profile of zebrafish exposed to Mn. Fish exposed to Mn for 96 h presented a significant reduction in the total distance traveled (F 4,80 = 22.15, P < 0.0001) (Fig. 1A), absolute turn angle (F 4,80 = 10.89, P < 0.0001) (Fig. 1B), time spent in top area (F 4,80 = 17.16, P < 0.0001) (Fig. 1C) and transitions to top area (F 4,80 = 16,65, P < 0.0001) (Fig. 1D) when compared to the control. These effects were elicited by all
Discussion
Excessive Mn is well known to be associated mainly with the development of motor and cognitive disorders (Harischandra et al., 2019, Taylor et al., 2020, Tuschl et al., 2013). Herein, we performed some neurobehavioral analyses in adult zebrafish to investigate the impact of environmental relevant short exposure to Mn on the appearance of psychiatric disturbances, especially anxiety-like phenotypes, referencing cell viability, AChE activity, cortisol and brain Mn levels as underlying biomarkers
Conclusion
In summary, we showed that in addition to locomotor deficits, short Mn exposure caused anxiety behavior in adult zebrafish. Our findings also indicated that brain AChE activity and cortisol dysregulation seem to follow Mn toxicity in adult zebrafish. However, we emphasize that additional mechanistic studies are needed to understand the involvement of cortisol and AChE system with the neurobehavioral changes found in the animals after Mn exposure.
CRediT authorship contribution statement
S.A.F. and N.V.B. designed the research. S.A.F., J.S.L, and M.M.S. performed the research. S.A.F., J.S.L, and M.M.S. analyzed the data. S.A.F. and N.V.B wrote the manuscript. All authors reviewed and edited the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The financial supports by FAPERGS, CAPES, and CNPq are gratefully acknowledged. N.V.B is a recipient of CNPq fellowships.
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