Characterization, effect of metal ions and organophosphates on the brain acetylcholinesterase of Nile Tilapia (Oreochromis niloticus) in vitro

Abstract

Acetylcholinesterase (AChE; EC 3.1.1.7) inhibition in vitro is an effective method for monitoring pesticide contamination, particularly organophosphates, offering advantages over in vivo approaches. Environmental factors like temperature, age, and stress can alter enzyme activity in vivo, making controlled in vitro studies necessary. This research focused on the brain AChE of Nile tilapia (Oreochromis niloticus), a key species in global aquaculture, to detect organophosphate contamination. The study examined pesticides such as chlorpyrifos, fenitrothion, malathion, and temephos. Optimal pH (8.0) and temperature (55°C) conditions were determined for AChE activity. The enzyme also showed sensitivity to several heavy metals, including Co²⁺, Cd²⁺, Cu²⁺, Hg²⁺, Mn²⁺, and Zn²⁺, with mercury causing 100% inhibition. AChE exhibited high substrate affinity, with a Vmax of 0.651 ± 0.26 mU/mg and a Michaelis-Menten constant (Km) of 399 mU/mg. Pesticide inhibition varied, with chlorpyrifos and temephos showing the highest efficacy at lower concentrations. IC₂₀, IC₅₀, and K_i values ranked pesticide potency as follows: malathion, fenitrothion, temephos, and chlorpyrifos. These results support the use of Nile tilapia AChE as a reliable environmental biomarker for detecting pesticides, offering valuable insights for environmental monitoring and filling gaps in the existing literature.

Key words
Characterization; ions; organophosphates; brains; acetylcholinesterase; nile

INTRODUCTION

Cholinesterases (ChEs) are widely used as biomarkers to diagnose the exposure of natural populations to organophosphates (OPs) and carbamate pesticides. Pesticides can be transported to aquatic ecosystems through spray drift, leaching, agricultural drainage, or surface runoff during precipitation or thaw events (Lewan et al. 2009, Desrochers et al. 2024). The most recent 2021 report published by the European Union (EU) on pesticide residues in food reveals that, out of 87,863 samples analyzed, 38,947 (44.3 percent) contained quantifiable concentrations of more than one pesticide. Furthermore, multiple residues were detected in 23,177 samples (26.4%), with a single sample of raisins containing up to 39 different pesticide residues.

With the widespread use of pesticides in agriculture and other industries, exposure to these harmful chemicals has become a growing concern, highlighting the need for safer and more sustainable pest control methods (Park et al. 2022, Rajagopalan et al. 2023). The residues of these compounds present in water and fish tissues affect cholinesterase activity, which remains inhibited for several weeks, making it a valuable tool in the environmental monitoring of OP pollution. These pesticides pose serious environmental problems due to their high neurotoxic effects, impacting both insects and non-target organisms such as fish, through acetylcholinesterase (AChE) inhibition (Albendín et al. 2017).

Although the residues of these compounds in water and fish tissues typically disappear within a few days, cholinesterase activity remains inhibited for several weeks, becoming a valuable tool in monitoring OP pollution. These pesticides pose serious environmental problems due to their highly neurotoxic effects, impacting both insects and non-target organisms such as fish, by inhibiting acetylcholinesterase (AChE) (Albendín et al. 2017).

AChE plays a crucial role in the nervous system by hydrolyzing the neurotransmitter acetylcholine (ACh) at cholinergic synapses, ensuring the intermittent nerve impulses necessary for neuronal communication (Lopes et al. 2019). The main enzymatic functions of AChE include deactivating ACh to prevent postsynaptic overstimulation, participating in neuronal tissue development, and hydrolyzing ghrelin, acting as a hydrolase (Brimijoin et al. 2016).

In fish, such as Nile tilapia (Oreochromis niloticus), there is considerable diversity in the biochemical properties and distribution of cholinesterases, as well as their sensitivity to anticholinesterase agents both in vitro and in vivo (Assis et al. 2011). This fish species is one of the leading species in global aquaculture, ranking third in production (4,407.2 thousand tons – 9% of the total volume), due to its rapid growth, high meat quality, and economic and social benefits (Nunes et al. 2024, Zhang et al. 2022).

Due to its ability to reflect responses to toxic substances, Nile tilapia is frequently used as a model in ecotoxicological studies to assess the effects of harmful substances on fish health (Hasan et al. 2022). However, there are few reports in the literature investigating the sensitivity of O. niloticus AChE in in vitro studies and the characterization of this enzyme. Therefore, this study aims to explore the potential use of Nile tilapia (Oreochromis niloticus) acetylcholinesterase as a biomarker, as well as to perform its physicochemical characterization and analyze the influence of metal ions on enzymatic activity.

MATERIALS AND METHODS

Preparation of crude AChE extract

The fish were obtained from fish farms in the city of São José de Ribamar, located in the metropolitan region of São Luís Island, Maranhão, Brazil. The fish were sacrificed by immersion in an ice bath before being acquired and were transported in thermal boxes with ice at 4°C to the Aquatic Organisms Biotechnology Laboratory at the Universidade Federal do Maranhão (UFMA).

The crude AChE extract was prepared according to De Carvalho Silva et al. (2024). The brains were immediately excised, pooled, and homogenized in a mechanical shaker at 5 rpm in a 0.5 M Tris-HCl buffer solution, pH 8.0, at 4°C, to prevent the loss of enzymatic activity, maintaining a ratio of 20 mg of tissue per mL. The homogenates were centrifuged for 10 minutes at 1000×g (4°C), and the supernatants, containing the crude extracts, were frozen at -30°C for subsequent assays.

Enzymatic activity of AChE

The activities of AChE were assessed using a modified version of the colorimetric method described by Assis et al. (2010). In a final volume of 240 μL, the following were added: 200 μL of DTNB (0.25 mM), and 20 μL of crude extract, and the reaction was initiated by the addition of 20 μL of acetylthiocholine iodide (62 mM). The enzymatic activity was determined by reading the increase in absorbance at 405 nm for 180 seconds at 25°C in a microplate reader. One unit of activity (U) was defined as the amount of enzyme capable of converting 1 μmol of substrate per minute.

Protein concentration

The protein concentration was estimated using the Warburg & Christian (1941) method on a spectrophotometer at absorbances of 260 nm and 280 nm using the following formula:

$$[\text{Protein concentration}]mg/mL=A260nm\times 1.55-A280nm\times 0.75$$

Physicochemical parameters

Optimal pH and stability

Different pH ranges were tested, varying from pH 3.0 to 9.0, using 0.1 M acetate buffer, 0.1 M Tris-HCl buffer, and 0.1 M citrate-phosphate buffer, with the reaction maintained for 3 minutes. To evaluate stability, the reaction at each pH range was maintained for 1 hour at 35°C, and its activity was analyzed. Relative activity was determined by taking the highest enzymatic activity at 100%.

Temperature and stability

To determine the optimal temperature, the crude acetylcholinesterase extract was maintained for 3 minutes at temperatures ranging from 20 to 80°C. To evaluate stability, the reaction was kept for 1 hour at each temperature, and its activity was analyzed. Relative activity was determined by taking the highest enzymatic activity at 100%.

Enzymatic kinetics

The Michaelis-Menten constants (K_m ) and the maximum hydrolysis rate (V_max ) were estimated using the methodologies described by Assis et al. (2012) and Dos Santos et al. (2022). Enzymatic activity was tested with increasing concentrations of the substrate acetylthiocholine (ASCh), ranging from 0.8 to 20.8 mMol/L (final concentration), for the estimation of K_m and V_max .

Influence of ions

To investigate the influence of ions on enzyme activity, we used the methodology described by Bocquené et al. (1990) with some adaptations. Acetylcholinesterase (AChE) was exposed to different salts containing ions (​Fe²⁺,​ ​Mg²⁺,​ ​Ca²⁺,​ ​Cu²⁺,​ ​Mn²⁺,​ ​Co²⁺,​ ​Zn²⁺,​ ​Cd²⁺,​ ​Hg²⁺​) at concentrations ranging from 0.001 to 10 mMol/L for 1 hour each. Relative activity was determined by taking the absence of inhibitor as 100%.

Inhibition assay with organophosphates

The AChE inhibition assays were conducted using the methodology of Lopez et al. (2019) with adaptations. The organophosphates temefos, malathion, fenitrothion, and chlorpyrifos were diluted in ultrapure water at concentrations of 0.0001, 0.01, 0.1, 0.5, and 1 ppm (Table I), with each subsequent concentration increased tenfold. The pesticide solutions (10 μL) were incubated with the crude extract (10 μL) for 1 hour at 25°C, and relative activity was determined by considering the activity in the absence of pesticide as 100%.

Table I
Concentrations of temephos, malathion, fenitrothion, and chlorpyrifos diluted in ultrapure water.

Compound	0.0001 ppm (μmol/L)	0.01 ppm (μmol/L)	0.1 ppm (μmol/L)	0.5 ppm (μmol/L)	1 ppm (μmol/L)
Temephos	0.0004665	0.004665	0.04665	0.0010716	0.0021432
Malathion	0.000000003027	0.00000003027	0.000330358	0.000165179	0.000330358
Fenitrothion	0.000000000003607	0.004665	0.000027723	0.0018045	0.00027723
Chlorpyrifos	0.000000000002853	0.004665	0.0002853	0.0014265	0.002853

Estimation of inhibition parameters IC₂₀, IC₅₀, and K_i

The concentrations that inhibit enzymatic activity by 20% and 50% (IC₂₀ and IC₅₀) were estimated for each pesticide according to Dos Santos et al. (2022), using the non-linear regression models obtained in the previous sections on the graphs of Ln (natural logarithm of the inhibitor concentration) vs. enzymatic activity (%) for each inhibitor. The K_i parameter (dissociation constant of the inhibitor-enzyme complex) was determined according to the equation proposed by Cheng & Prusoff (1973):

$$Ki\hspace{0.25em}=\frac{I{C}_{50}}{1\hspace{0.25em}+\frac{[S]}{K_m}}$$

Where:

IC50 = concentration capable of inhibiting 50% of enzymatic activity;

[S] = represents substrate concentration;

Km = Michaelis-Menten constant.

Statistical analyses

The results of this study were analyzed using ANOVA and Tukey’s analysis (p < 0.05) with GraphPad Prism® software version 8.0.1. The results were reported as mean ± standard deviation (SD) of triplicate determinations.

RESULTS AND DISCUSSION

Optimal pH and stability

The maximum activity and pH stability of acetylcholinesterase (AChE) from O. niloticus were achieved at pH 8.0 in a 0.1 mM Tris-HCl buffer solution (Figure 1a and b). Furthermore, AChE demonstrated significant activity in other pH ranges, maintaining activity above 40%, which evidences its resistance to pH variations (Figure 1a).

Figure 1
Optimal pH (a) and Stability (b) of AChE from O. niloticus. Data expressed as relative activity ± standard deviation. The activity was determined in each sample using acetylthiocholine iodide as substrate at 25°C for 3 minutes to determine the optimal pH and for 1 hour to determine stability. Triplicate tests (n = 5).

The investigation and characterization of the physicochemical parameters of cerebral AChE are crucial for establishing baseline activity values and identifying it as a suitable tool for environmental and food screening (Assis et al. 2012). AChE from O. niloticus exhibited an optimal pH and pH stability estimated at 8.0, while the enzymatic activity was not less than 55% in the pH range of 3 to 9.0 in both assays (Figure 1a and b). Similar results were found in studies of AChE present in crude extracts, such as Marinho et al. (2019), which obtained a pH of 9.0 with Danio rerio, C. undecimalis exhibiting an optimal pH of 8.0 (Lopes et al. 2019), Hoplosternum littorale with pH 8.0 (Araujo et al. 2018), Parachromis managuensis pH 8.0 (de Araújo et al. 2016), and Pleuronectes platessa with pH 8.5 (Bocquené et al. 1990).

AChE maintained its maximum activity after 1 hour at pH 8.0, in addition to demonstrating activity in other pH ranges, as reported by Lopes et al. (2019) and Assis et al. (2011). This wide distribution and resistance to different pH levels may be associated with migratory habits, and studies have reported that the optimal pH range for AChE varies between 6.5 and 8.5, depending on the species.

Optimal temperature and stability

The optimal temperature for AChE from O. niloticus was identified as 55°C (Figure 2a). The enzyme demonstrated good activity in the temperature range of 20 to 55°C, maintaining its relative activity above 40%. However, its activity decreased drastically above 80°C, with a loss of activity exceeding 70%. AChE from O. niloticus exhibited stability up to 55°C, indicating its ability to maintain activity at high temperatures.

Figure 2
Optimal Temperature (a) and Stability (b) of AChE from O. niloticus. Data expressed as relative activity ± standard deviation. The activity was determined in each sample using acetylthiocholine iodide as substrate at 25°C for 3 minutes to determine the optimal temperature and for 1 hour to determine stability. Triplicate tests (n = 5).

AChE from O. niloticus exhibited an optimal temperature of 55 °C and stability up to 55 °C after 1 hour (Figure 2a and b). This reflects the high tolerance of this fish to environmental variations, which influences its adaptive physiological characteristics, including the optimal pH and temperature of the enzymes involved in its metabolism (Ajima et al. 2017, Abou-Zeid et al. 2021). Other species also demonstrated similar results, such as Arapaima gigas, with an optimal temperature of 45 °C, and Raquicentron canadum, with 35 °C (Assis et al. 2012).

Kinetic parameters

The kinetic parameters, maximum hydrolysis rate (Vmax), and Michaelis-Menten constant (Km) were analyzed using the substrates ASCh. The Vmax values found for acetylcholinesterase from O. niloticus were 0.651 ± 0.26 mU/mg (nmol/min/mg of protein). The Michaelis-Menten constants (Km), which represent the substrate affinity for the enzyme, were 399 mU/mg (nmol/min/mg of protein). The values of these parameters for various fish species are shown in Table II.

Table II
Values of maximum velocities (V _max) and Michaelis-Menten constant (K _m) of AChE and physicochemical parameters of AChE from some freshwater and marine species.

Species	Km [mM]	Vmax [U/mg protein]	Optimum pH	Optimum temperature [°C]	Source	References
O. niloticus*	0.651 ± 0.36	399 ± 0.17	7.5	55	Brain
O. niloticus	0.39 ± 0.2	0.218 ± 0.007	8.0	35	Brain	Assis et al. (2012)
O. niloticus	0.10 ± 0.03	0.229 ± 0.014	-	-	Brain	Rodríguez-Fuentes & Gold-Bouchot (2004)
Centropomus undecimalis	0.99 ± 0.31	289 ± 13.13	8.0	45	Brain	Lopes et al. (2019)
Danio rerio	0.191 ± 0,024	0,677 ± 0,039	9.0	30	Brain	Marinho et al. (2019)
Diapterus auratus	0.49	215	7.2 – 8.0	35	Brain	Lopes et al. (2019)
Genyatremus luteus	0.153	-	7.2	25	Brain	De Carvalho Silva et al. (2024)
Colossoma macropomum	0.43 ± 0.02	0.129 ± 0.05	7.5 - 8.0	45	Brain	Assis et al. (2010)

• Data expressed as mean ± standard deviation; assays conducted in triplicate (n = 5). *Present study.

The results of this study revealed a greater affinity for the substrate, as well as higher V_max and K_m values compared to those found by Assis et al. (2012) and Rodríguez-Fuentes & Gold-Bouchot (2004), who also studied the same enzyme in O. niloticus. This disparity may be attributed to genetic or environmental factors; tilapias from different geographical regions or raised under distinct conditions may exhibit variations in enzymatic activity.

Influence of metal ions

Based on the obtained results (Figure 3), it was observed that among the ions present in the analyzed salts, only ​Fe²⁺​, at a concentration of 10 mMol/L, showed a significant increase in the enzymatic activity of AChE from O. niloticus, with an increase of 72.15%. On the other hand, ​Mg²⁺​ and​ Ca²⁺​ ions did not demonstrate a significant effect, with reductions in activity of 30.54% and 39.55%, respectively, at the highest concentration tested (10 mMol/L), indicating a strong inhibition of AChE from O. niloticus by these ions.

Figure 3
Influence of ions on the activity of AChE from O. niloticus. AChE was incubated with ions in 50 mM Tris-HCl at pH 8.0 for 1 hour at 25°C before determining relative activity. Triplicate tests were conducted, and the data are expressed as residual activity ± standard deviation.

On the other hand, the ions ​Co²⁺, Cd²⁺, Cu²⁺, Hg²⁺, Mn²⁺, and Zn²⁺​ exhibited a more substantial reduction, with inhibitions of 77.92% for Co²⁺, 71.84% for Cd²⁺, 69% for ​Cu²⁺​, 75.25% for ​Mn²⁺​, and 79.88% at a concentration of 10 mMol/L. For ​Zn²⁺​, there was a decrease in enzymatic activity at the lowest concentration (0.001 mMol/L), showing a reduction of 63.85%, which sharply increased to 100% inhibition at 10 mMol/L.

The increased activity of acetylcholinesterase (AChE) in O. niloticus when incubated with Fe²⁺ can be explained by several possible mechanisms, including modulation of enzymatic activity, alterations in oxidative stress, and cellular compensatory mechanisms. A study on fish exposure to sublethal concentrations of copper revealed an increase in specific activity and catalytic efficiency of AChE, suggesting that metal ions like Fe²⁺ may similarly affect AChE through metal homeostasis mechanisms or structural modifications of the enzyme (Romani et al. 2003).

Several studies have reported AChE activity inhibition by mercury, identifying it as the strongest inhibitor in other aquatic organisms (Atlin 2018, Silva et al. 2013, Assis et al. 2015, Araújo et al. 2016). The reduction of acetylcholinesterase (AChE) activity in O. niloticus when incubated with Hg²⁺ may be explained by the formation of complexes with sulfhydryl groups, disruption of metal ion homeostasis, or oxidative stress. Mercury (​Hg²⁺​) is a heavy metal known for its strong interaction with sulfhydryl (-SH) groups present in many enzymatic proteins, including AChE. This interaction can lead to conformational changes in the enzyme that result in inhibition of its activity (Frasco & Guilhermino 2007, Ajsuvakova et al. 2020).

These mechanisms explain how exposure to Hg²⁺ can lead to AChE inhibition in fish, serving as a concerning indicator of environmental contamination by heavy metals. The reduction of acetylcholinesterase (AChE) activity in O. niloticus when incubated with Zn²⁺ may be related to the interaction of this metal ion with the enzyme, including a competitive inhibition mechanism. Araújo et al. (2018) studied the effect of metal ions, including ​Zn²⁺​, on AChE in benthic fish and confirmed enzymatic inhibition, suggesting a competitive or allosteric inhibition mechanism.

The reduction of acetylcholinesterase (AChE) activity in O. niloticus when incubated with ​Co²⁺​ can primarily be explained by the direct interaction of cobalt with the enzyme, possibly acting as an inhibitor. In studies on the competitive inhibition of AChE, certain ions have been shown to directly compete for the active site of the enzyme. These studies generally explore the interaction of different ligands with AChE, providing insights into how ions such as ​Co²⁺​ may act similarly (Liu et al. 2017).

The reduction of acetylcholinesterase (AChE) activity in O. niloticus when incubated with Cd²⁺ can be explained by several mechanisms based on the toxic properties of cadmium, such as direct AChE inhibition, oxidative stress, and alterations in enzymatic conformation (Jebali et al. 2006). This study investigated AChE inhibition by cadmium in Seriola dumerili, highlighting a significant inhibition of enzymatic activity at elevated concentrations of this heavy metal (Araújo et al. 2018). The characterization of AChE in benthic fish and the analysis of the in vitro effects of various metal ions, including ​Cd²⁺​, suggest a direct interaction of the metal with the enzyme.

The ability of this enzyme to be less inhibited by ions such as ​Mg²⁺, Ca²⁺,​ and ​Cu²⁺​ compared to other species in the literature, along with the cost-benefit considerations of working with unpurified enzymes, may make the use of AChE from O. niloticus advantageous as a biomarker.

Effects of pesticides

The brain activity of AChE from O. niloticus decreased significantly in the presence of the studied organophosphates (Figure 4). At the lowest concentration (0.0001 ppm), there was a significant reduction of 63.84% in AChE activity when exposed to chlorpyrifos and fenitrothion, which increased sharply to 100% inhibition starting from a concentration of 0.1 ppm, with an inhibition of 49.54%.

Figure 4
Influence of pesticides on the activity of lipase F2 of AChE from O. niloticus. AChE was incubated with the pesticides for 1 hour at 25°C before determining residual activity. Triplicate tests were conducted, and the results are presented as residual activity ± standard deviation.

In contrast, when exposed to malathion and temephos, enzymatic activity showed a significant decrease starting at a concentration of 0.01 ppm. With temephos, there was a 29.44% inhibition at this concentration, increasing to 93.74% at the highest concentration (1 ppm). For malathion, a significant reduction in enzymatic activity was observed at a concentration of 0.01 ppm, with 58.05% inhibition, which increased to 60.74% at the highest concentration (1 ppm).

The results obtained from the effects of the four tested organophosphates (OPs) demonstrated that AChE from O. niloticus exhibited high sensitivity, surpassing the inhibition rate of 50% for all tested pesticides (Figure 4). As described by Araújo et al. (2018), a 20% inhibition of AChE activity suggests the presence of an anti-AChE agent, while a 50% inhibition indicates the onset of associated signs and symptoms, and a 90% inhibition often results in the death of the organism.

Therefore, the IC20 and IC50 parameters (the concentrations of pesticides that inhibit 20% and 50% of AChE activity, respectively) are essential, as they represent these situations and demonstrate the toxicity of a given compound in its interaction with AChE. AChE from O. niloticus was inhibited by the pesticides at concentrations lower than those permitted by the World Health Organization (WHO), which are 0.9 mg/L or 2.72 µmol/L for malathion in drinking water and 0.03 mg/L or 0.0856 µmol/L for chlorpyrifos in drinking water.

The estimated values for the inhibitory action (IC50) of brain AChE activity from O. niloticus were 1.068 µmol/L for chlorpyrifos, 5.167 µmol/L for fenitrothion, 3.076 µmol/L for malathion, and 1.099 µmol/L for temephos. Chlorpyrifos and fenitrothion produced the highest IC50 values among the tested organophosphates. The values of IC20, IC50, and Ki for these pesticides are presented in Table III.

Table III
Values of IC50, IC20, and Ki for in vitro AChE from O. niloticus in the analysis of pesticides.

Pesticide	IC20 (μmol/L)	IC50 (μmol/L)	Ki (μmol/L)
Chlorpyrifos	0.42	1.068	0.132
Fenitrothion	2.07	5.167	0.998
Malathion	123.4	3.076	0.138
Temephos	0.439	1.099	0.898

The IC₅₀ values found for AChE from O. niloticus in relation to the studied pesticides, using IC20 as a criterion, revealed a decreasing order of inhibitory potency among organophosphates: malathion, fenitrothion, temephos, and chlorpyrifos (Table III). It was observed that the pesticide chlorpyrifos and temephos showed relatively low IC₂₀, IC₅₀, and K_i results, demonstrating high efficacy in inhibiting AChE from O. niloticus. Fenitrothion exhibited moderately low results, indicating considerable efficacy in inhibiting the enzyme. The results of this study are similar to those of Chauhan et al. (2016) with the pesticide chlorpyrifos, where the acetylcholinesterase of the electric eel is inhibited at concentrations of 0.1 - 60 nMol/L, showing the significant sensitivity of brain AChE from fish to this pesticide.

On the other hand, malathion presented higher IC₂₀ values compared to the other pesticides studied, suggesting lower efficacy in inhibiting AChE at lower concentrations. However, the IC₅₀ value was lower compared to other pesticides, indicating reasonable efficacy at higher concentrations, as also observed in Figure 4. Some pesticide compounds, such as malathion, may have lower toxicity due to efficient detoxification mechanisms (Hodgson & Rose 2005, Rajagopalan et al. 2023).

Although pesticide residues typically undergo a series of degradation processes in nature, they can persist in the ecosystem for a considerable period due to their greater stability and reduced water solubility (Székács & Darvas 2015). This affects marine organisms like fish, which are of great interest to the food industry, thereby impacting on the quality of products offered in the market. For this reason, the inhibition of cholinesterases has been widely used as a biomarker for detecting contamination in aquatic environments, due to the sensitivity of these enzymes to the toxic effects of various contaminants, such as organophosphate pesticides and insecticides (De Carvalho Silva et al. 2024).

Other studies show the potential use of brain AChE from commercially significant fish as potential biomarkers for these contaminants, such as Centropomus undecimalis and Colossoma macropomum. The inhibition of this enzyme is widely employed for the detection of organophosphates. Furthermore, it is important to highlight that these enzymes can also be affected by other contaminant agents, including heavy metals, as demonstrated in this study. In the context of ecotoxicology, these findings reinforce the relevance of using this enzyme as a valuable tool in environmental monitoring and in assessing the quality of fish offered in the market, thereby reducing the risks associated with exposure to contaminants.

CONCLUSIONS

The cerebral acetylcholinesterase (AChE) of O. niloticus demonstrated sensitivity to various heavy metals and pesticides, including ​Co²⁺, Cd²⁺, Cu²⁺, Hg²⁺, Mn²⁺, and Zn²⁺,​ particularly to mercury (​Hg²⁺​). The pesticides chlorpyrifos, fenitrothion, malathion, and temephos also resulted in significant inhibitions of AChE from O. niloticus, with chlorpyrifos and fenitrothion being notably effective at lower concentrations. Few studies to date have investigated the in vitro response of O. niloticus brain AChE to these pesticides, highlighting its potential use as a biomarker for these anticholinesterase compounds in environmental monitoring. Since the fish collected for this study were obtained from a farming operation, there is a possibility of variability that may or may not be directly connected to natural environments, necessitating more detailed studies.

ACKNOWLEDGMENTS

The authors would like to thank the Universidade Federal do Maranhão (UFMA), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES: Funding code 001: PROCAD Amazon number 23038015702/2018 – M.D.S.B.N), and the Fundação de Amparo à Pesquisa e ao Desenvolvimento Tecnológico do Maranhão (FAPEMA: doctoral scholarship BD-02367/23 – A.K.C.S) for their financial support.

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Publication Dates

• Publication in this collection
  31 Mar 2025
• Date of issue
  2025

History

• Received
  25 June 2024
• Accepted
  9 Jan 2025