Changes in Enzyme Activities in the Plasma of Tilapia guineensis Exposed to Glyphosate in the Laboratory

Eze BU, Ogolo C, Okenwa U and Nwosu P

Published on: 2024-07-26

Abstract

Changes in the activity of enzymes are useful markers of stress brought on by any toxin that fish have been exposed to. Decreases in intestinal microflora activity and immunological responses may be the outcome of their activity disturbances. The present study examined the effects of various glyphosate concentrations (0.00, 0.50, 1.00, 1.50, and 2.00 mg/l) on the activities of enzymes in the plasma of Tilapia guineensis exposed for a period of 15 days, including aspartate transaminase (AST), alanine transaminase (ALT), acid phosphatase (ACP), alkaline phosphates (ALP), and lactate dehydrogenase (LDH). The analysis of the enzyme study's results showed that every enzyme significantly (P <0.05) exceeded the control values. The juvenile fish exposed to the alteration had a greater degree of severity than the adult fish, depending on the concentration. In conclusion, changes in plasma enzyme parameters can be used as rapid and reliable indicators of monitoring toward the effects of toxicants on aquatic organisms and ultimately the ecosystem as a whole. These parameters may be attributed to target tissue damage and dysfunction brought on by toxicants.

Keywords

Toxicants; Aquatic environment; Herbicides; Tilapia; Enzymes

Introduction

One of the main global environmental problems confronting humanity in the recent past is aquatic pollution [1]. Many rivers in the nation are dealing with complex pollution issues as a result of industry and the haphazard development that is common in the nation's largest cities [2,3]. Because of them, the ecosystem has been dangerously contaminated and degraded, especially the aquatic environment [4]. In a living thing, enzymes are crucial for metabolism and food use [5]. Herbicides have been identified as one of the primary contaminants of aquatic ecosystems that can negatively affect living creatures in the short- or long-term. It appears that they have caused several metabolic alterations in fish, some of which are deadly and others of which are more commonly sublethal. Stress and pesticide effects can affect hormones and enzymes, which can be dangerous for fish species. Fish populations are more significantly impacted by chronic low level exposure than by acute poisoning. Pesticide concentrations not high enough to kill fish are linked to subtle changes in physiology and behavior that hinder fish survival and reproduction [6]. On the other hand, alterations in fish ion concentrations, organic components, enzyme activity, endocrine activity, and chemo-regulators have been linked to pesticides [7].

Serum enzyme activity measurements are widely used as a diagnostic technique in human medicine [8]. The use of enzyme activity as a predictor of pesticide toxicity in aquatic animals, especially fish, has increased significantly in recent years [9]. Amino transferases, phosphatases, and lactases are among the most important group of enzymes found in lower animals, especially telost fish [10]. The interconversion of a keto acid into an amino acid is catalyzed by a group of enzymes known as transferases, which also includes alanine transaminase (ALT) and aspartate transaminase (AST). Although phosphatases are hydrolase enzymes, acid phosphatase (ACP) and alkaline phosphatase (ALP) are the ones that eliminate phosphate groups from a range of substances, such as proteins, alkaloids, and nucleotides [11]. The enzyme lactase, also known as lactate dehydrogenase, is also involved in the process of converting muscle-derived lactic acid into pyruvic acid, which is a prerequisite for the synthesis of cellular energy [12]. When fish are exposed to toxins, which are known to interfere with the activities of enzymes, they go through physiological modifications to preserve equilibrium [13]. Fish with altered protein metabolism have been exposed to most toxicants for extended periods of time [14]. The reduction in total protein in fish exposed to dangerous levels of toxicant may be caused by either a disruption in liver protein synthesis or a change in the fish's water equilibrium and state of hydration, or maybe both [15]. Every biological activity is governed by proteins called hormones and enzymes.Protein and enzyme activities might be used as a diagnostic tool to assess the physiological state of cells or tissues [16].

Enzymes are essential for an animal's food digestion and maintenance of its metabolic functions. They are extremely particular about the type of reaction they catalyze and the substrate they use, making them incredibly efficient. The membrane permeability of cells was affected by pollutants, xenobiotics, or toxicants present in the waterbodies as macromolecules. These substances may also bind to enzymes, modify the quantities of cofactors or reactants, or have an indirect effect on enzyme activity [18]. Fish exposed to industrial effluent experience altered activity of hydrolytic enzymes such as transminases and esterases due to its high conductivity and low dissolved oxygen content [19].In toxicological research, enzymes are sensitive biomarkers because they can reveal early warning signs of potentially dangerous alterations in aquatic species that are suppressed in contaminated water [20]. Fish physiology can sustain relative damage from long-term exposure to toxins, and this damage can be estimated using metabolic enzymes, which are employed as clinical indicators [21]. Enzymes are also helpful biomarkers for evaluating in-vivo environmental exposures in fish [22]. Fish abnormalities can be found by measuring enzymes such LDH, AST, ALP, ACP, and ALP [23].Thus, the current study aims to evaluate the changes in the activity of the following enzymes in the plasma of Tilapia guineensis exposed to glyphosate in a lab setting: lactate dehydrogenase, acid phosphatase, alanine transaminase, and aspartate transaminase.

Materials and Methods

Experimental Location and Fish

The study was carried out at the Nigerian Institute for Oceanography and Marine Research's branch office, the African Regional Aquaculture Center, in Buguma, Rivers State. Ponds produced 360 T. guineensis during low tide, of which 180 were juveniles and 180 adults. Six 50-liter open plastic containers containing the fish were delivered to the lab, where they were allowed to adapt for seven days.

Preparation of Test Solutions and Exposure of Fish

Glyphosate was used in this experiment; it was purchased from a supermarket in Port Harcourt, Nigeria. Two sizes of T. guineemsis were exposed to the material in triplicate at concentrations of 0.50, 1.00, 1.50, and 2.00 mg/L, with a control of 0.00. Ten fish were randomly arranged in each test tank. Fifteen days were dedicated to the test. Fresh water was added to the tanks each day. Commercial feed was fed to the fish twice a day at a body weight of 3%.

Analytical procedure

At the end of each experimental session, a tiny needle was used to puncture the caudal artery to obtain a 2 ml sample of fresh blood, which was then transferred into vials that had been heparinized.Blood samples were centrifuged for 15 minutes at 5000 rpm right away.Before being examined, separated plasma samples were pipetted into eppendorf tubes and stored in a freezer at -20°C [24]. The data was read using a Jenway visible spectrophotometer (Model 6405) equipped with a universal microplate reader. Five enzymes were measured in the blood of the exposed T. guineensis: lactate dehydrogenase (LDH), aspartate amino transaminase (AST), alanine amino transaminase (ALT), alkaline phosphatase (ALP), and acid phosphatase (ACP). The Reitman and Frankel [25] method was used to investigate AST since it may be completed manually using a colorimetric end-point technique. While the Huang et al. [26] method was used to carry out ALP, ACP, and LDH.

Statistical Analysis

The mean and standard deviation of the mean were used to express all the data. The data analysis was done using SPSS Version 22, a statistical program. Using two-way ANOVA, the means were split, and the two means were deemed significant at 5% (P <0.05).

Results

With the exception of DO, where lower values were found at greater chemical concentrations, the water quality metrics (Table 1) were all within the same range. Table 2 shows the effects of glyphosate on the enzymes in the plasma of juvenile T. guineensis. It was shown that as the herbicide concentration increased, the values of SOD and GSH dropped. On the other hand, CAT and LPO considerably rose in comparison to the control values. The antioxidant levels of adult fish exposed to the toxin showed a similar pattern (Table 3).

Table1: Physico-Chemical Parameters of Water in Experimental Tanks of T. guineensis Exposed to Glyphosate.

Concentrations (mg/L) DO  (mg/L) Temperature  (oC) pH NH3  (mg/L)   Salinity (ppt)
0 5.89±0.03 b 29.88±2.01a 6.64±0.66 a 0.01±0.00 a 11.32±0.89 a
0.5 5.71±0.43b 29.81±3.01 a 6.67±0.34 a 0.02±0.00 a 11.33±0.32 a
1 5.06±0.88 b 29.74±1.01 a 6.66±0.33 a 0.02±0.00 a 11.76±1.89 a
1.5 4.77±0.33 a 29.82±5.02 a 6.64±0.81 a 0.03±0.00 b 11.71±0.55 a
2 4.07±0.44a 29.84±4.00 a 6.61±0.91a 0.03±0.00 b 11.88±1.82 a

Means within the same column with different super scripts are significantly different (P<0.05)

Table 2: Enzymes Activities in T. guineensis Juveniles Exposed to Glyphosate.

Concentrations (mg/L) Enzymes (IU/L)
AST ALT ACP ALP LDH
0 58.22±1.04a 45.03±1.77 a 15.02±1.87 a 50.55±2.77 a 215.03±8.01 a
0.5 62.02±1.04 a 48.08±1.02 a 17.56±1.05 a 55.72±1.55 a 235.03±9.42 a
1 70.02±3.66 b 57.00±1.08 b 21.82±1.33 b 65.77±2.57 b 260.02±4.55 b
1.5 75.33±3.25 b 70.01±1.54 c 25.54±1.66 b 74.55±1.88 c 279.02±3.77 b
2 85.02±3.00 c 75.01±1.07 c 30.66±2.53 b 80.44±2.49 d 299.44±9.44 b

Means within the same column with different super scripts are significantly different (P<0.05)

Table 3: Enzymes Activities in T. guineensis Adults Exposed to Glyphosate.

Concentrations (mg/L) Enzymes (IU/L)
AST ALT ACP ALP LDH
0 72.04±1.43 a 53.33±1.59 a 15.12±1.03 b 61.45±7.55 a 320.04±9.01 a
0.5 80.81±7.03 b 58.78±1.07 a 19.46±1.02 a 64.71±4.99 a 340.44±9.77 a
1 84.23±2.89 b 65.41±1.73 b 24.88±1.01 b 70.02±9.65 b 370.72±9.43 b
1.5 89.12±3.45 b 70.88±1.86 c 30.89±1.87 c 73.02±4.77 b 381.99±7.01 b
2 98.07±3.34 c 75.22±1.61 c 34.01±1.34 c 80.02±9.03 c 393.01±9.52 b

Means within the same column with different super scripts are significantly different (P<0.05)

Discussion

ACP and ALP activity are useful markers of toxicant stress in fish. Any harm or malfunction in the test organs is a sign that the activity of phosphatase has changed. The study reports an increase in T. guineensis plasma exposed to different concentrations of glyphosate. An increase in the activity of acid and alkaline phosphatases can be explained by the tissue shifting its focus from the regular ATPase system to the phosphatase system for energy breakdown. Pesticides raise phosphorylate activities while lowering glycogen levels [27]. Activated phosphatases can catalyze the release of inorganic phosphatases from phosphate esters, which can lead to phosphorylation in the case of a reduced ATPase system. Herbicide poisoning changed the ACP and ALP's activity in the current study. The lysosomal enzyme ACP exhibits an increase in activity in response to cellular injury [28]. An accelerated rate of enzyme turnover under pesticide stress may be the cause of the elevated ACP and ALP activity. These phosphatases, also known as phosphomonoesterases, are active at particular pH values. The duration, pesticide value, and concentration all affect how ACP and ALP alter. These two enzymes, ACP and ALP, were elevated in fish Labeo rohita as a result of alterations brought about by pesticide dichlorvos toxicity; however, the elevation of ALP is far less significant than that of ACP [29].

Animal abnormalities are typically discovered in laboratories by detecting different enzymes, such as AST, ALT, and LDH [30]. Since the non-plasma specific enzymes ALT, AST, and LDH are

found in the tissue cells of the liver, heart, gills, kidneys, muscles, and other organs, their presence in the blood may provide particular information regarding organ dysfunction. Diverse studies have reported differences in the activity of these enzymes as a result of the impact of pollutants or toxins on different fish organs in different species [31]. Many soluble enzymes found in blood serum have been suggested as helpful stress markers. Fish illness diagnosis and environmental contamination-related tissue damage can be determined by measuring serum ALT, AST, and LDH activities. Stress-related tissue deterioration is indicated by an increase in these enzyme activity in the serum or extracellular fluid, which is a sensitive indicator of even mild cellular damage [32].

The plasma of T. guineensis exposed to glyphosate had increased levels of all the enzymes evaluated in this experiment. This observation aligns with the results of Gabriel et al. [33], who observed elevated serum ALT, AST, and LDH activity in African catfish (Clarias gariepinus) treated to cypermethrin. The fish Rhamdia quelen [34] and Labeo rohita [35] showed a considerable rise in plasma ALT, AST, and LDH activities following their exposure to cypermethrin. Sub-acute dosages of deltamethrin and pyrethroid were given to Nile tilapia for a period of 28 days, and this too caused a spike in serum ALP [36]. These researchers concluded that hepatic necrosis and the consequent leakage of this enzyme into the bloodstream may be the cause of the spike in this enzyme in the blood.Because of the way that toxicants affect hepatocytes, increased ALT, AST, and LDH levels usually indicate liver degeneration and hypofunction. This is because the release of cellular enzymes into the blood plasma is caused by the destruction of tissue. Consequently, the hepatotoxic effect of toxicants is demonstrated by the fact that increases in these enzyme activities in T. guineensis serum are mostly brought about by the leaking of these enzymes from the liver cytosol into the blood stream as a result of liver damage brought on by metals and pesticides [37]. Elevated blood levels of these enzymes are usually indicative of illness and liver necrosis in animals, according to Tamas et al. [38]. Cellular damage in the liver may cause blood levels of ALT, AST, and LDH to increase.

Conclusion

Sub-lethal glyphosate concentrations can cause enzymatic breakdown and other toxicological consequences in T. guineensis juveniles and adult sizes. Blood biochemical profiles can provide important insights into an organism's internal environment since the blood is the first organ to be impacted by adverse environmental changes. The current study's findings supported the theory that changes in fish exposed to pesticides' plasma enzyme activity were a biochemical manifestation of the adverse effects of toxicants. Fish treated with pesticides exhibited larger increases in all plasma enzyme parameters compared to the control group that was not exposed to any pesticides. In conclusion, changes in plasma enzyme parameters can be used as rapid and precise indicators of monitoring toward the impact of toxicants on aquatic organisms and ultimately the ecosystem as a whole. These parameters can be attributed to target tissue damage and dysfunction caused by toxicants.

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