The tissue-specific nature of physiological zebrafish mitochondrial bioenergetics.

Zebrafish are a powerful tool to study a myriad of experimental conditions, including mitochondrial bioenergetics. Considering that mitochondria are different in many aspects depending on the tissue evaluated, in the zebrafish model there is still a lack of this investigation. Especially for juvenile zebrafish. In the present study, we examined whether different tissues from zebrafish juveniles show mitochondrial density- and tissue-specificity comparing brain, liver, heart, and skeletal muscle (SM). The liver and brain complex IV showed the highest O2 consumption of all ETC in all tissues (10x when compared to other respiratory complexes). The liver showed a higher potential for ROS generation. In this way, the brain and liver showed more susceptibility to O2- generation when compared to other tissues. Regarding Ca2+ transport, the brain showed greater capacity for Ca2+ uptake and the liver presented low Ca2+ uptake capacity. The liver and brain were more susceptible to producing NO. The enzymes SOD and Catalase showed high activity in the brain, whereas GPx showed higher activity in the liver and CS in the SM. TEM reveals, as expected, a physiological diverse mitochondrial morphology. The essential differences between zebrafish tissues investigated probably reflect how the mitochondria play a diverse role in systemic homeostasis. This feature may not be limited to normal metabolic functions but also to stress conditions. In summary, mitochondrial bioenergetics in zebrafish juvenile permeabilized tissues showed a tissue-specificity and a useful tool to investigate conditions of redox system imbalance, mainly in the liver and brain.

Using structural equation modeling to assess the genotoxic and mutagenic effects of heavy metal contamination in the freshwater ecosystems: A study involving Oreochromis niloticus in an urban river.

Chemical pollutants represent a leading problem for aquatic ecosystems, as they can induce genetic, biochemical, and physiological changes in the species of these ecosystems, thus compromising their adaptability and survival. The Capibaribe River runs through the state of Pernambuco, located in Northeastern Brazil, and passes through areas of agricultural cultivation, densely populated cities, and industrial centers, primarily textiles. Despite its importance, few ecotoxicological studies have been conducted on its environment, and knowledge about pollution patterns and their effects on its biota is still being determined. The objective of this study was to evaluate the water quality and the damage supposed to be caused by pollutants on the DNA specimens of Nile tilapia (Oreochromis niloticus) obtained from seven strategic points of Capibaribe. Tilapia specimens and water were collected during the rainy and dry seasons from 2015 to 2017. The following characteristics were analyzed: physicochemical (six), metal concentration (seven), local pluviosity, micronuclei, and comet assay. The physicochemical and heavy metal analyses were exploratory, whereas the ecotoxicological analyses were hypothetical. To verify this hypothesis, we compared the groups of fish collected to the results of the micronuclei test and comet assay. We created a Structural Equation Model (SEM) to determine how each metal's micronuclei variables, damage index, pluviosity, and concentration were related. Our results demonstrated that the highest values for markers of genetic damage were detected at points with the highest heavy metal concentrations, especially iron, zinc, manganese, chromium, and cadmium. The SEM demonstrated that metals could explain the findings of the genotoxicity markers. Moreover, other pollutants, such as pesticides, should be considered, mainly where the river passes through rural areas. The results presented here demonstrate that the Capibaribe River has different degrees of contamination and confirm our hypothesis.

Characterization of brain acetylcholinesterase of bentonic fish Hoplosternum littorale: Perspectives of application in pesticides and metal ions biomonitoring.

Acetylcholinesterase (AChE; EC 3.1.1.7) is a serine hydrolase, whose main function is to modulate neurotransmission at cholinergic synapses. It is, therefore, the primary target of some pesticides and heavy metals. Its inhibition in aquatic organisms has been used as an indicator of the presence of these pollutants in water bodies. The present study aimed to characterize physicochemical and kinetic parameters of brain AChE in the benthic fish Hoplosternum littorale and to analyze the in vitro effects of pesticides (dichlorvos, diazinon, chlorpyrifos, parathion-methyl, temephos, carbaryl, carbofuran, aldicarb, diflubenzuron, novaluron and pyriproxyfen) and metal ions (As3+, Cd2+, Cu2+, Fe2+, Mn2+, Mg2+, K+, Pb2+, Hg2+, Zn2+) investigating the potential of this enzyme as environmental biomarker based on current regulations. Specific substrates and inhibitors have indicated AChE to be the predominant cholinesterase (ChE) in the brain of H. littorale. Peak activity was observed at pH 8.0 and 30 °C. The enzymatic activity is otherwise moderately thermostable (≈ 50% activity at 45 °C). The enzyme can reduce the activation energy of acetylthiocholine hydrolysis reaction to 8.34 kcal mol-1 while reaching a rate enhancement of 106. Among the pesticides under study, dichlorvos presented an IC50 value below the maximum concentrations allowed by legislation. This study presents the first report on the inhibition of brain AChE activity from Siluriformes by the pesticides novaluron and pyriproxyfen. Mercury ion also exerted a strong inhibitory effect on its enzymatic activity. The H. littorale enzyme thus has the potential to function as an in vitro biomarker for the presence of the pesticide dichlorvos as well as mercury in areas of mining and industrial discharge.

Acetylcholinesterase of mangrove oyster Crassostrea rhizophorae: A highly thermostable enzyme with promising features for estuarine biomonitoring.

Enzyme biomarkers from several aquatic organisms have been used for assessing the exposure to contaminants at sublethal levels. Amongst them, the cholinesterases are commonly extracted from several organisms to evaluate/measure organophosphate and carbamate neurotoxic effects. Acetylcholinesterase (AChE; EC 3.1.1.7) is an enzyme of the group of serine esterases that acts on the hydrolysis of the neurotransmitter acetylcholine allowing the intermittence of the nerve impulses responsible for the neuronal communication. This enzyme is the main target for the action of some pesticides and the inhibition of its activity in bivalve mollusks may be used as biomarker due to their filter-feeding habit. In this context, the present study aimed to characterize physicochemical and kinetic parameters of the AChE extracted from gills and viscera of the oyster Crassostrea rhizophorae and investigate the in vitro effect of pesticides (dichlorvos, diazinon, chlorpyrifos, methyl-parathion, temephos, carbaryl, carbofuran, aldicarb, diflubenzuron and novaluron) in search for assessing its potential as biomarker. Specific substrates and inhibitors evidenced the predominance of AChE in both tissues. The optimum pH found for gills and viscera AChE were 8.0 and 8.5, respectively. The maximum peak of activity occurred at 70 °C for gill AChE and 75 °C for viscera AChE. The enzymes of both tissues presented remarkable thermostability. The Michaelis-Menten constant for both enzymes were 1.32 ±â€¯0.20 mM for gills and 0.43 ±â€¯0.12 mM for viscera. The Vmax values for gills and viscera were 53.57 ±â€¯1.72 and 27.71 ±â€¯1.15 mU/mg, respectively. The enzymes were able to reduce the activation energy to 9.75 kcal mol-1 (gills) and 11.87 kcal mol-1 (viscera) obtaining rate enhancements of 3.57 × 105 and 1.01 × 104, respectively, in relation to non-catalyzed reactions. Among the pesticides under study, the carbamates carbaryl and carbofuran exerted the strongest inhibitory effects on the enzyme activity achieving important degrees of inhibition at concentrations below national and international current regulations. The first observation of the effects of benzoylurea pesticides (diflubenzuron and novaluron) on AChE from mollusks is reported here. The gills AChE of C. rhizophorae showed potential to be specific biomarker for the carbamate carbaryl while the viscera AChE showed it for carbofuran. According to their features, these enzymes may be proposed as promising tools for estuarine monitoring as well as biocomponent of biosensor devices.

Brain acetylcholinesterase of jaguar cichlid (Parachromis managuensis): From physicochemical and kinetic properties to its potential as biomarker of pesticides and metal ions.

This contribution aimed to characterize physicochemical and kinetic parameters of the brain cholinesterases (ChEs) from Parachromis managuensis and investigate the in vitro effects of pesticides and metal ions on its activity intending to propose as biomarker. This species is suitable for this investigation because (1) it was recently introduced in Brazil becoming invasive (no restrictions on capture) and (2) occupies the top of the food chain (being subject to bioaccumulation). The enzyme extract was exposed to 10 metal ions (Al(3+), Ba(2+), Cd(2+), Cu(2+), Hg(2+), Mg(2+), Mn(2+), Pb(2+), Fe(2+) and Zn(2+)) and ChEs selective inhibitors (BW284c51, Iso-OMPA, neostigmine and serine). The extract was also incubated with organophosphate (dichlorvos) and carbamate pesticides (carbaryl and carbofuran). Inhibition parameters (IC20, IC50 and ki) were determined. Selective inhibitors and kinetic parameters confirmed acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) -like as responsible for the ChE activities, most AChE. The IC50 values for pesticides were: 1.68µM (dichlorvos); 4.35µM (carbaryl) and 0.28µM (carbofuran). Most of the analyzed ions did not show significant effect at 1mM (p=0.05), whereas the following ions inhibited the enzyme activity in the order: Hg(2+)>Cu(2+)>Cd(2+)>Zn(2+). Mercury ion strongly inhibited the enzyme activity (IC20=0.7µM). The results about allow to conclude that P. managuensis brain AChE is a potential biomarker for heavy metals and pesticides under study, mainly for the carbamate carbofuran once it was capable to detect 6-fold lower levels than the limit concentration internationally recommended.