American Journal of Biochemistry and Molecular Biology

Year: 2017 | Volume: 7 | Issue: 1 | Page No.: 21-40
DOI: 10.3923/ajbmb.2017.21.40

Atrazine-induced Changes in some Biochemical Parameters of the Early Developmental Stages of the African Catfish Clarias gariepinus (Burchell, 1822)

Imam A.A. Mekkawy , U.M. Mahmoud and Sara Salah

Abstract: Background and Objective: Atrazine is considered the most common aquatic herbicide used. So, the present study aimed at studying atrazine-induced changes in some biochemical characteristics during early embryonic stages of Clarias gariepinus . These characteristics include two metabolic enzymes, lactate hydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH), two oxidative enzymes, glutathione reductase (GSH) and superoxide dismutase (SOD), two thyroid hormones, triiodothyronine (T3) and thyroxine (T4), lipid peroxidation (LPO), DNA damage and patterns of protein fractions of different early developmental stages. Material and Methods: Gametes were collected from mature specimens of the African catfish, C. gariepinus from the river Nile. The fertilized eggs were incubated in dechlorinized tap water (pH = 7.7, dissolved oxygen 88-94% saturation, temperature 27-29EC and photoperiod 12:12 (light:dark). Under atrazine influences, different measurements of enzyme activities, electrophoretic protein analysis, lipid peroxidation, DNA fragmentation, Total Protein (TP) and hormones were carried out. Results: The atrazine-induced significant fluctuation in the activity of G6PDH and LDH with a sharp decrease in 24 h-PFS after hatching. There is no significant variations in GSH activity under atrazine doses except in 24 and 96 h-PFS with a decrease in all developmental stages except at 72 h-PFS. Insignificant fluctuation in SOD activity in all developmental stages of C. gariepinus except 4, 20 and 24 h-PFS in comparison to control. Atrazine induced significant fluctuation in DNA damage and LPO/TP ratio in all developmental stages. Insignificant fluctuation in T3 activity but significant decrease in T4 activity in all developmental stages of C. gariepinus under exposure of sublethal doses of atrazine. Conclusion: One could conclude that atrazine significantly disturbe the metabolic parameters of embryonic and developmental stages of fishes and in turn most of these parameters could be used as indicators of environmental aquatic pollution.

Keywords: hormones, electrophoresis, Atrazine, Clarias, biochemistry and development

INTRODUCTION

In Egypt, waste water and agriculture drains containing pesticides are discharged directly into rivers and lakes from industrial plans, aerial spraying of field and water drainage system¹. These pesticides are stored under conditions that rarely meet internationally accepted standards² leading to magnification of their contamination problem. Previous studies^3-10 have reported that the exposure of fish to pesticides resulted in several changes many biochemical parameters including enzyme systems and hormones.

All chemical reactions in the cell are catalyzed by enzymes and controlled by hormones especially in embryonic and adult stages^11,12. Introduction of foreign chemicals and pesticides in the cell generally disturb many enzyme and hormone functions^13-20. Atrazine is one of the most common and significant pesticides that belongs to the s-triazine family of herbicides.

The natural antioxidant activity in animal body is a potential mechanism which contributes to the prevention of a variety of oxidative damages, toxicity and other diseases^21-23. The antioxidant defense has become an important aspect of investigation in mammals and animals^24,25. In this concern, fish are used as bio-indicators because the effects of exposure to xenobiotics can be measured in terms of biochemical or physiological responses²⁶. Changes in age and species distribution in a fish population are also general indicators of water pollution but there are also responses specific to a single pollutant or a group of xenobiotics²⁷.

African catfish Clarias gariepinus widely distributed throughout Africa²⁸ has become one of the most important fishes used as bioindicator in terms of its biochemical and physiological characters^9,28-36. Early developmental stages are considered to be one of the most sensitive stages in the fish life cycle to the toxic effects of chemical contaminants^29,33,37-41.

Accordingly, the present study aimed at studying atrazine-induced changes in some biochemical characteristics during early embryonic stages of Clarias gariepinus to discover new discover environmental bioindicators for atrazine pollution . These characteristics include 2 metabolic enzymes, lactate hydrogenase and glucose-6-phosphate dehydrogenase, 2 oxidative enzymes, glutathione reductase and superoxide dismutase, 2 thyroid hormones, triiodothyronine (T3) and thyroxine (T4), lipid peroxidation, DNA damage and patterns of protein fractions of different early developmental stages.

MATERIALS AND METHODS

Gamete collection: Mature specimens of the African catfish, C. gariepinus (weight of 900-1500 g) were collected from the river Nile at Assuit, Egypt (during spawning seasons from April-July) and transported to the Fish Lab., Zoology Department, Assuit University.

The criteria applied for the selection of spawners were those described by De Graaf and Janssen⁴². For collection of semen, male fishes were anaesthetized with 200 mg L^–1 Ms_222 (tricaine methane sulphonate, Crescent Research Chemicals, Phoenix, Arizona, USA) buffered with 800 mg L^–1 sodium bicarbonate and one of the testes was removed surgically. Alternatively the fish were killed and the whole gonads were removed. Testes were cleaned from the blood by surgical towels. The sperms from the testes were pressed through a mesh fabric into a sterile dry petri dish and used directly for dry fertilization after ensuring the healthy and motility of sperms. For collection of eggs, ovulation was artificially induced. Females were injected intra-peritoneal with pellets (gonadotropin-releasing hormone analogue, GnRHa, D-Ala6, Pro9 Net) containing 2.5-3.0 mg of water soluble dopamine antagonist metoclopramide (Interfish Ltd., Hungary) dissolved in 0.65% NaCl. One pellet was used per kg body weight. After injection (10-11 h), the fish were stripped and the eggs were collectedin clean dry plastic containers, dry fertilization was considered.

Source of atrazine: A stock solution of herbicide atrazine (90% purity) provided by syngenta, Bazal, Switzerland (commercial name: Gesaprim 90 WG) was prepared by dissolving 1.10 g of the substance in 1 L of dechlorinated water.

Experimental setup and sampling: The fertilized eggs were incubated in dechlorinized tap water (pH = 7.7, dissolved oxygen 88-94% saturation, temperature 27-29°C and photoperiod 12:12 (light:dark). Exposure started 4 h after fertilization (4 h-PFS) then at intervals, 16, 24, 48, 72, 96 and 120 h-PFS for triiodothyronine (T3), thyroxine (T4), Glutathione Reductase (GR) and superoxide dismutase (SOD) in one experiment. The embryos were divided into 4 groups: One control and three concentrations 9, 18 and 27 mg L^–1 of commercial herbicide atrazine. All working solutions were made immediately prior to use. Exposure took place in 12 petridishes (14 cm in diameter and 2.5 cm high), 3 petri dish replicates for each group (treated water level was 2 cm) for each developmental stage.

In another experiment, exposure started 4 h after fertilization then at intervals, 8, 12, 16 and 24 h-PFS for lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PDH), lipid peroxidation (LPO) and DNA damage using three sublethal doses of atrazine (9, 18 and 27 mg L^–1) in comparison to the control. The fertilized eggs were divided into eight groups: One control and seven concentrations 0.1, 1, 5, 10, 20, 100 and 200 mg L^–1 of commercial herbicide atrazine. Exposure took place in 24 petri dishes (14 cm in diameter and 2.5 cm high), 3 petri dish replicates for each group (treated water level was 2 cm) for each developmental stage.

Measurements of enzyme activities: Because of the small size of the embryos, whole body homogenates were used for the measurements of the enzyme activities³⁷. Homogenization of the whole animal is a reliable method that provides a reasonable index of total enzyme activity in individuals whose body mass is predominantly composed of muscle tissue^43,44. The embryos were pulverized under liquid nitrogen and 100 mg of ground tissue powder was added to 2 volumes of buffer [50 mM tris, pH 7.4, 1 mM ethylene diamine tetra-acetic acid (EDTA) and 2 mM Mg Cl₂]. Tissue was homogenized briefly with an Ultra-T URRAX (temperature was maintained at 4°C during homogenization). The homogenate was centrifuged for 15 min at 10,000xg and 4°C and the supernatant was used for the enzyme activity assay. Activities were determined in the supernatant with a spectrophotometer (Micro Lab 200 Vital Scientific, Dieren, The Netherlands) at a wavelength of 340 nm and at 37°C using kits, Stanbio LDH (UV-Rate) procedure No. 0940 USA for the quantitative determination of lactate dehydrogenase⁴⁵ and RANDOX Laboratories Ltd., PD410, UK BT294QY for the quantitative determination of glucose-6-phosphate dehydrogenase⁴⁶ G6PDH. The calculation of the catalytic activity content for the selected enzymes was in accordance with the recommendations of the French Society of Clinical Biology⁴⁷. The principle of the assay consists of the kinetic determination of LDH activity, based on the rate of NADH oxidation. We determined the oxidation rate, which is directly proportional to LDH activity by measuring the decrease in absorbance at 340 nm. To standardize the expressions of enzyme activity, the units of enzyme activity have been defined as the quantity of enzyme that catalyzes the reaction of 1 mmol of substrate min^–1. The catalytic concentration was expressed as U L^–1.

In addition to determine the antioxidant enzymes at wavelength 340 nm and 37°C using kits, biodiagnostic (UV method) for the determination of glutathione reductase⁴⁸ which is essential for the glutathione redox cycle that maintains adequate levels of reduced cellular GSH. The GSH serves as an antioxidant, reacting with free radicals and organic peroxides, in amino acid transport and as asubstrate for the glutathione peroxidases and glutathione S-transferases in the detoxification of organic peroxide and metabolism of xenobiotics,respectively. And at wavelength 560 nm for the determination of superoxide dismutase⁴⁹ which is metalloenzyme that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and thus form acrucial part of the cellular antioxidant defense mechanism.

Protein analysis by polyacrylamide gel electrophoresis (SDS–PAGE): Embryos (~0.1 g fresh weight) of each treatment in addition to control were suspended in 1.0 mL lysing buffer, heated at 100°C for 5 min centrifuged at 10,000 rpm for 30 min and 50 μL of each extracted protein treatment was used for protein analysis using SDS-PAGE according to Laemmli⁵⁰ in the first dimension. The low-molecular weight standards (Pierce, USA) were run concurrently and the protein molecular mass was determined using Gel-Pro analyzer package version 3.1 for Windows XP/NT (Media Cybernetica 1993-97).

Lipid peroxidation and total protein measurements: Total protein contents were determined according to the Biuret method⁵¹ using bovine serum albumin (E. Merck-Darmstadt, Germany) as a standard. Lipid peroxidation (LPO) in the embryos was determined by the procedure of Utley et al.⁵². The absorbance of each aliquot was measured at 535 nm. The rate of lipid peroxidation was expressed as nmol of thiobarbituric acid reactive substance (TBARS) formed per h per milligram of protein using a molar extinction coefficient⁵³ of 1.56 M^–1 cm^–1.

DNA fragmentation measurement: The DNA fragmentation was determined by the procedure of Kurita-Ochiai et al.⁵⁴ using spectrophotometer (Micro lab 200 vital scientific Dieren the Netherlands) at 575 or 600 nm against reagent blank. The percentage of fragmented DNA was estimated by the following formula:

Hormones measurements: According to the method of Cobb et al.⁵⁵, the triiodothyronine (T3) and total thyroxine (T4) in embryos after homogenated in phosphate buffer saline were measured according to the method of Skelley et al.⁵⁶ using ELISA kits (with Cat# BC-1005 and BC-1007 for T3 and T4, respectively). These kits were obtained from BioCheck, Inc. 323 Vintage Park Dr., Foster city, CA94404.

Statistical analysis: The basic statistics, means, standard errors and ranges were estimated. The patterns of variation due to developmental stages and atrazine doses and their interaction were studied by two way analysis of variance using the SPSS package⁵⁷ at the 0.05 significance level. Levene’s test of equality of error variance of the dependent variables was applied, with rejection of the null hypothesis for raw, log-transformed and SQRT-transformed data. So, the homogeneity of variance was assumed for raw data. The Tukey-HSD test was considered for multiple comparisons. Moreover, the Dunnett, t-test was applied, measuring the control against other treatments in each developmental stage.

RESULTS

Metabolic enzymes: G6PDH and LDH: The atrazine doses have different adverse impacts on 2 metabolic enzymes (G6PDH and LDH) during early embryonic development of Clarias gariepinus. The atrazine-induced activity of G6PDH (Table 1, Fig. 1) was fluctuated significantly with developmental progress (p<0.001) with a peak in 12 h-PFS and a sharp decrease in 24 h-PFS directly after hatching. Such sharp decrease may refer to the fact that zygotic gene of G6PDH is activated during this later stage. Similarly, significant fluctuation (p<0.001) in G6PDH activity was evident under atrazine doses with no special trends towards decrease or increase in each developmental stage studied.

The atrazine-induced activity of LDH (Table 2, Fig. 2) was fluctuated significantly in some cases and increased in others with developmental progress (p<0.001) with a sharp decrease in 24 h-PFS directly after hatching. Such sharp decrease may refer to the fact that zygotic gene of LDH is activated during this later stage.

Similarly, significant fluctuation (p<0.001) in LDH activity was evident under atrazine doses with no special trends towards decrease or increase in each developmental stage studied. However, LDH activity increased in higher atrazine doses and in old stages studies (Table 2) till 24 h-PFS.

Oxidative enzymes: GR and SOD: In each of the developmental stages studies (4-120 h-PFS) of C. gariepinus, there is no significant variations in GR activity under atrazine sublethal doses except in 24 and 96 h-PFS stages (Table 3). Such variations (p>0.05) were directed towards decrease in all developmental stages except 72 h-PFS which show fluctuation in enzyme activity under stress. Except for the control group (p<0.05), the GR activity exhibited insignificant variations among developmental stages.

Atrazine-induced SOD activity insignificantly (p>0.05) fluctuated in all developmental stages of C. gariepinus except 4, 20 and 24 h-PFS in comparison to the control. Except for the control and 27 mg L^–1 groups (p<0.05), the SOD activity exhibited insignificant variations among developmental stages under the stress of other sublethal doses (Table 4).

DNA damage: Atrazine-induced DNA damage significantly (p<0.05) fluctuated in all developmental stages of C. gariepinus except 4 h-PFS in comparison to the control. Except for the control, 0.1 and 10 mg L^–1 groups (p>0.05), the DNA damage exhibited significant variations among developmental stages under the stress of other sublethal and lethal doses (Table 5) (p<0.0001). Significant interaction between atrazine doses and embryonic stages was evident.

Lipid peroxidation relative to total protein (LPO/TP): Atrazine-induced LPO/TP significantly (p<0.0001) fluctuated in all developmental stages of C. gariepinus. The LPO/TP exhibited also significant variations among developmental stages under the stress of sublethal and lethal doses (Table 6) (p<0.0001). Significant interaction between atrazine doses and embryonic stages was evident.

Thyroid hormones: T3 and T4: Atrazine-induced T3 variations insignificantly (p>0.05) fluctuated in all developmental stages of C. gariepinus. Except for the control, 0.1 and 27 mg L^–1 groups (p<0.0001), T3 activity exhibited insignificant variations among developmental stages under the stress of other sublethal doses (Table 7) (p>0.05).

No significant impacts of atrazine sublethal doses on the activity of T3. The variations in the T3 activity may be present in the treated group as well as the control one. Only in the older early embryonic stages (72 and 120 h-PFS), the atrazine sublethal doses led to significant decreased change in T4 activity in comparison to the control (Table 8). Except for 9 mg L^–1, within each treatment, T4 activity exhibited significant variations among developmental stages. Such variations may not be related to atrazine doses but to the stage of development.

Protein electrophoresis: The protein fractions of different developmental (4, 8, 12, 16, 20, 24, 48, 72, 96 and 120 h) stages used in the present study were identified in terms of their molecular weight and concentration under atrazine (0.1, 1, 5, 9, 10, 18, 20, 27, 100 and 200 mg L^–1 ) stress in comparison with the control. Variability in these 2 parameters and number of bands were evident reflecting variations in protein gene expression under atrazine stress, developmental stages and their interactions (Table 9-12).

DISCUSSION

The contamination of aquatic ecosystems by xenobiotics has gained increasing attention^58-62 and several recent studies have demonstrated the toxicity and effects to fish under field and laboratory conditions^4,63-69. Atrazine is one of the most widely used herbicides and because of its considerable persistence and mobility in soil and water, it is considered a common terrestrial and aquatic contaminant⁷⁰. Atrazine treatment increased hydrogen peroxide (H₂O₂) content, while reducing gene expression and enzymatic activities related to two major H₂O₂-detoxification pathways⁷¹.

The uptake kinetic of atrazine exhibited a three-compartment characteristic. It had already reached the embryo within 10s of exposure time and is rapidly absorbed in the first 6 h, then followed steady state conditions between 6 and 24 h. Atrazine uptake is also influenced by elimination process since such process was more effective between 24 and 48 h⁷². In the present study, such pattern of atrazine uptake was evident by its adverse fluctuated effect on different biochemical parameters considered.

The activities of enzymes such as LDH and G6PDH have been used as bioindicator of stress in catfish Clarias garepinus^{9,29,32,33,73}. The variations in these two metabolic enzyme systems of the embryonic stages of Ctenopharyngodon idellus and Clarias gariepinus were studied by Mekkawy and Lashein⁴⁰, Mekkawy et al.⁷⁴ and Osman et al.³³. These researchers discussed the developmental time of activation and inactivation of the maternal and zygotic genes through those early developmental stages. The switching time (i.e., blastula, gastrula and organogenesis) from maternal to zygotic LDH and G6PDH-genes was determined in the control and stressed developmental stages. The pattern of LDH and G6PDH activities in the present study was also determined directly before 24 h-PFS. Interaction between atrazine doses and developmental stages was evident with fluctuating variations in the enzyme activities. Decrease in G6PDH activities under pesticide stress is well documented in literature^75,76. Both the decreased concentration of G6PDH and the increased activity of LDH can be explained as a consequence of stress-based especially hepatic tissue⁷⁷. The SOD and GSH provide the first line of defense against oxygen derived free radicals^4,78-80. Different studies deals with these enzymes under different types of stress^81-86 especially herbicides^3-5 in fishes. Increase in the SOD activities is probably a response towards increased ROS generation in pesticide toxicity⁸⁷. Generally exposure of animals to xenobiotics increases SOD activity in various tissues^87,88. The St. Dimitrova et al.⁸⁹ reported a simultaneous induction response in SOD activities in carp Cyprinus carpio exposed to zinc. Different researchers referred to increase^90-92 or decrease^76,93-96 in GSH activities under stress in fishes. Fluctuations GSH and SOD activities under atrazine stress was evident in most developmental stages with interaction between atrazine doses and progress in early development. The pesticide toxicity results from the metabolic conversion processed in the endoplasmic reticulum of cells in concern directly affecting the morphology of these cells^97,98. Oxidative stress may be due to the depletion of GSH which prevents the conjugation of xenobiotics like atrazine to GSH and thus enables them to freely combined covalently with cell proteins⁹⁹. Jayachandran et al.⁹⁰ reported that the reduced levels of GSH in the atrazine administered Labeo rohita could be the result of either decreased utilization of GSH for induced toxicity. Envionmental stress has been demonstrated to cause an increase in the oxidative stress, an imbalance in the antioxidant status¹⁰⁰. Nammalwar¹⁰¹ reported that the change in glutathione redox ratio and hence a decrease in reduced GSH levels, the glutathione-mediated detoxification process may also be affected. This might be a factor responsible for the lack of elimination of toxic compounds that enter the fish and thus result in their accumulation, aggravating oxidative stress. Yildirim et al.⁷⁸ concluded that a decline in tissue GSH content during exposure to pollution may be due to an increased utilization of GSH, which can be converted into oxidized glutathione and in-efficient GSH regeneration. However, severe oxidative stress may suppress GSH levels due to the impairment of adaptive mechanisms¹⁰².

Exposure of fishes to pesticides and other xenobiotics is known to induce lipid peroxidation in various tissues especially liver ones, which is responsible for the adverse biological effects^103-105. Previous investigations have reported the induction of LPO by pesticides such as deltamethrin^106,107, alachlor¹⁰⁸, malathion¹⁰⁹ and butachlor⁵ in fish. Parthasarathy and Joseph⁹³ demonstrated that λ-cyhalothrin as a type of pesticides might have stimulated lipid peroxidation by influencing a variety of reactions in freshwater tilapia Oreochromis mossambicus. Nwani et al.⁴ also reported that the elevated level of lipid peroxidation in the liver of Channa punctatus in response to the exposure to atrazine resulting from reactive oxygen species which may lead to cell apoptosis.

The degree of lipid peroxidation is associated with toxic doses of chemicals, time of exposure, species, developmental stages and their interactions^{4,10,33,40,74}. Most of these findings were evident in the present study by Atrazine-induced changes in LPO in early embryonic stages of C. gariepinus. Singh et al.¹¹⁰ reported that reactive oxygen species, such as superoxide anions, hydroxyl radical and hydrogen peroxide enhance the oxidative process and induce peroxidative damage to membrane lipids. So, the fluctuation in current pattern of LPO may be one of the molecular and adaptation mechanisms involved in the atrazine-induced toxicity on early embryonic stages.

Different investigations emphasized on DNA damage under chemical stresses in fishes^29,33. Many of these investigations have shown that ROS is the major source of DNA damage by causing strand breaks, removal of nucleotides and various modifications of the nucleotide bases¹¹¹. Variability of DNA damage were found to be related to species, age, chemical doses and time of their exposure and their interactions. In the present study, pattern of variations in DNA damage was evident in concern with the early embryonic stages of C. gariepinus and atrazine doses.

The sensitivity of thyroid gland to many toxicants have gained much attention and a number of endocrine disrupting compounds have been^30,112. The thyroid hormones are known for their osmoregulatory and metabolic effects^113-115 and are involved in the regulatory act of stress tolerance in fish^116-118. These hormones also control development, growth and reproduction of fishes^{113,117,119-128}. It is possible that the elevated plasma levels of thyroid hormones in broodstock during reproduction are necessary to ensure an adequate concentration of hormones in the developing oocyte^127,129. Moreover, several studies have demonstrated the beneficial effects of increased levels of maternal T3 on subsequent larval development and survival, e.g., striped sea bass and the rabbitfish Siganus guttatus^128,130. In many species studied, there is a clear evidence that the concentration of T4 and T3 decreases as embryogenesis proceeds. The biological significance of such evidence is still unclear^131,132.

Different pesticides have been found to change the pattern of secretion of thyroid hormones in adult and embryonic stages of fishes^30,133,134. Exposure of catfish Heteropneutes fossilis and Clarias batrachus to malathion and endosulfan caused changes in circulatory thyroid hormones^133,134. A decrease in T3 has been reported in rainbow trout exposed to acidic water¹³⁵ and starvation^136-140. A change in T3 and T4 hormones have been reported in adult catfish Clarias gariepinus exposed to 4-nonylphenol⁴¹. In the present study, fluctuations in T3 and T4 activities in the developmental stages of C. gariepinus were insignificant in most cases and referred to the fact that it may not be related to atrazine doses but to the stage of development.

In the present study, the Variability in the molecular weight, concentration and number of bands were evident reflecting variations in protein gene expression under atrazine stress, developmental stages of C. gariepinus and their interactions. Our results are consistent with those of Mekkawy et al.²⁹ who found that the protein fractions in fish embryos exposed to UV-A have fluctuations in their protein concentration and their molecular weight in comparison with control embryos.

CONCLUSION

It is concluded that atrazine induces oxidative stress, DNA damage, lipid peroxidation and endocrine disruption on the developmental stages of African catfish Clarias gariepinus. Most of these parameters could be used as indicators of environmental aquatic pollution. However, fluctuations in T3 and T4 activities in the developmental stages of C. gariepinus were insignificant in most cases and referred to the fact that it may not be related to atrazine doses but to the stage of development.

SIGNIFICANCE STATEMENT

Discovering new indicators to assess environmental risks is an important issue especially with the increase of use of herbicides like atrazine. So this study and the related others are a must especially in concern with hormone disruptors and genotoxicity. The sensitivity of such situations is related to the chemicals as well as fish species and the aquatic communities.

REFERENCES