Toxic Effects of Triazophos on Metabolism in Fish.

frequently seeps into river waters and fish farming ponds that are river-fed. Consequently, these rivers along with nearby fish farming ponds are at a high risk of pesticide contamination.

Marine organisms living in contaminated waters often build up significant amounts of numerous chemicals, even when the surrounding environment's pollution levels are not high (Colombo et al. 1995). This can present a potential danger to the entire food chain. As a toxic substance enters a lifeform, it triggers biochemical and physiological reactions, which may either be adaptive or result in harmful consequences. The biochemical processes are typically the foremost and delicate indicators of harm caused by pollution. Therefore, it's crucial that we identify and explain the impacts of pollutants in biochemical terms, to understand how they operate and possibly find ways to alleviate their harmful effects.

Research has been conducted in India to explore the biochemical impacts of pesticides on water-dwelling creatures (Begum and Vijayaraghavan, 1999; Bhavan and Geraldine, 2001; Tripathi and Singh, 2002). Nevertheless, a substantial knowledge gap persists regarding the enduring effects of pesticide exposure. As an extension of this study, we evaluated levels of critical metabolites and enzymes in liver and muscle tissues of Channa punctatus (Bloch) fish during contact with commercial triazophos (O, O - diethyl, 0.1 phenyl, 1H, 1, 2, 4 ,Triazol 3yl – phosphorothio), as well as post-contact. C. punctatus is highly sought after for consumption purposes and often reared in paddy fields using the "rice-and-fish" farming method in India which exposes them to insecticides over prolonged periods regularly. The examined metabolites and enzymes participate in essential biological processes including protein synthesis and carbohydrate metabolism.

Methods and Material Details

C. punctatus fish were procured

from Nirmal Lake, situated in the district of Adilabad, Andhra Pradesh, with an average weight of around (50±1.30g) and a mean length measurement of (25.5±1.21 cm). These specimens were maintained in glass tanks filled with 60L of dechlorinated water, sourced from the top layer of the lake and given an unrestricted diet which included commercial fish food and boiled beef liver cut into small pieces. A three-week acclimatization period in a laboratory environment maintained at a constant temperature of 22±1.0°C was ensured before any experiments were conducted. The water inside the tanks was replaced once every 24 hours. The IUPAC nomination for triazophos pesticide is (O, O - diethyl, 0.1 phenyl, 1H, 1, 2, 4, Triazol 3yl - phosphorothio). As reported by Bayne and his co-researchers (1977), the LC50 (48h) value which represents the lethal concentration of triazophos for C. panctatus was found to be 0.019 ppm.

For each concentration, six vessels were set up, and every vessel contained 6 fish in 6 liters of tap water that had been dechlorinated. Throughout the experiment, the water's temperature was kept stable at 22±1.0°C. The quality parameters of the test water such as PH=7.2-7.3; dissolved oxygen=9.2 mg/lit; free carbon dioxide=10.0 mg/lit; and alkalinity=69 mg per liter, were measured using the APHA/WEF (1998) method. There was no additional treatment for the control groups' housing water which was also dechlorinated tap water. No food was given to the fish for a whole day before or during the experiment duration. After periods of 24, 48, 72 and 96 hours exposure time, all fish were removed from their respective containers and washed with freshwater before being euthanized by a strong hit on

their heads.

The research involved the study, extraction, and use of liver and muscle tissues to determine metabolite and enzyme levels. The metabolites assessed were total protein, free amino acids, ammonia, and glycogen. Enzymes such as alanine aminotransaminase (E.C. 2.6.1.2) (ALAT), aspartate aminotransaminase (E.C. 2.6.1.1) (AAT), and glutamate dehydrogenase (?-glutamate-NAD(P)-oxidoreductase, E.C. 1.4.1.3) were also scrutinized in this study.
Total protein was evaluated using Lowry et al.'s method from 1951 whereas the method by Moor and Stein from 1954 was employed for quantifying amino acids.
Ammonia concentration was determined via Bergmeyer's approach from1965 utilizing Nessler reagent; similarly Klicepera et al.'s technique from 1957 was used for measuring glycogen amounts.
Enzymes like ALAT & AAT were gauged using Rietman & Frankel's procedure referenced by Bergmeyer in his writings during 1965.
Finally, Lee and Lardy's methodology introduced in 1965 facilitated the assessment of glutamate dehydrogenase.

Enzyme activity evaluation was conducted using 5% (w/v) homogenates from liver and muscle tissues. These were processed in a homogenizer equipped with an ice jacket and filled with 0.25 M frozen sucrose solution, utilizing a motor-powered pestle unless otherwise mentioned below. The homogenates were then spun in a chilled centrifuge at 3000 x g for a span of 20 minutes to remove nuclei and cellular debris. Following this, the clear cell-free extracts that emerged were employed to measure enzyme activities. Enzyme activity quantification was performed spectrophotometrically, employing appropriate enzyme and reagent blanks at either 30 or 35?C.

The ideal settings for each enzyme analysis are detailed below: The Alanine aminotransaminase assay necessitates a 1 ml concoction consisting of 100 µmol DL-alanine (pH 7.4), 2 µmol ?-ketoglutaric acid (pH 7.4), and 100 µmol phosphate buffer (pH 7.4). This mixture is combined

with an enzyme source created in a sucrose solution (0.25M) amounting to approximately 0.2 ml, which is then left to incubate for half an hour. Similarly, the Aspartate aminotransaminase test requires the same volume containing identical amounts of L-aspartic acid (pH 7.4), ?-ketoglutaric acid (pH 7.4), and phosphate buffer (pH 7.4). An enzyme source, prepared in an equivalent concentration of sucrose solution as previously mentioned(0.25 M), is also incorporated into this blend. After this procedure, Tussaky and Shorr's method from1953 is employed to quantify the released inorganic phosphate.

The enzyme activity of glutamate dehydrogenase was tested under specific conditions. A 2 ml preparation, with ingredients like phosphate buffer of 100 µmol and pH 7.4, 75 µmol of sodium glutamate, 2 µmol INT (2,4-iodophenyl-3-(4-nitropheny)5-phenyltetrazolium chloride), 0.1 µmol NAD, and 0.5 ml of enzyme source made in a sucrose solution (0.25M), was used for the assay. This reaction mixture, additionally with a volume of 2 ml, was then allowed to incubate for a duration of half an hour.

Findings and Analysis

The data about total protein, amino acids, ammonia and glycogen are displayed in Table 1. The total protein content in the liver saw a noticeable reduction during treatment with triazophos. A similar trend was also observed in muscle tissue where the protein content dropped after 24h, 48h, 72h, and 96hrs of exposure. Throughout the exposure time and even after the triazophos exposure was withdrawn, liver amino acid content was largely stable. A rise in amino acid content was largely seen during 24h, 48h, 72h, and 96hrs of exposure in the muscle tissue. The triazophos exposure led to increased ammonia content in both liver and muscle of C. punctatus. In

terms of glycogen, its level significantly reduced in liver tissue at 24h, 48h, 72h, and 96hrs intervals of exposure. Conversely, only a minor decrease was noticed in the muscle tissue's glycogen content throughout the triazophos treatment.

Triazophos had a detrimental impact on the glutamate dehydrogenase in both liver and muscle tissues, as detailed in Table 2. An apparent rise in ALAT activity was observed in the liver after 24 hours, 48 hours, 72 hours, and 96 hours exposure to triazophos. Such increase was also noticeable in muscle ALAT activity following triazophos treatment. Additionally, the Aspartate aminotransaminase activity witnessed a surge in the liver of fish subjected to triazophos, when compared to those unexposed. Similar increment in AAT activity was also noted in muscle tissue during exposure to triazophos (Refer to Table 2).

The activity of Liver GDH was elevated at the 24h, 48h, 72h, and 96h intervals during exposure to toxicants. Although there was also a rise in the GDH activity in muscle tissue during these periods, it was less dramatic compared to that observed in the liver.

The exposure led to a noticeable reduction in liver protein content but had less significant changes in muscle tissue protein content, like the observed decrease in liver and muscle protein content in Heteropneustes fossilis fish exposed to pesticides documented by Singh et al. (1996), and in C. batrachus fish exposed to carbofuran according to Singh and Sharma (1998). The protein content drop in the organs of exposed fish signifies the physiological adaptability of fish, potentially in response to exposure-related stress (Vega et al. 2002). During triazophos exposure, free amino acids seemed to fluctuate more in muscles than the liver,

possibly utilizing them for energy synthesis and other metabolic processes (Shobha Rani and Janaiah, 1991). Despite the changes, the influence on free amino acids was inconsistent and generally marginal.

There were higher ammonia levels in the liver and muscle tissue of fish exposed for a particular duration than in those of control fish during all observation days. This aligns with previous observations in C. batrachus exposed to trichlorfon (Shobha Rani and Janaiah, 1991). The increased ammonia concentration could be due to amplified ammoniogenesis in the liver and muscle tissues. Ammonia is a harmful by-product, and an excess quantity is likely to initiate detoxification or utilization systems, primarily via the generation of less harmful nitrogen-based compounds like urea and glutamine (Krebs, 1980). While urea and glutamine were not assessed in this current study, such a mechanism may have been at play in C. punctatus.

The current research revealed that triazophos significantly reduced glycogen concentration in the liver and to a lesser extent in the muscle. Earlier research implies that such changes can escalate the levels of both catecholamines and glucocorticoids in the bloodstream (Nakano and Tomlinson, 1967). Consequently, the significant glycogenolysis noticed following the immediate exposure to triazophos in this study was likely a result of stress-induced boosting of circulating catecholamines and glucocorticoids. The liver, being the main organ for storing glycogen, exhibited a more pronounced decrease than the muscle.

Activities of Alanine and aspartate transaminase could be employed as sensitive indicators in teleost fish during experimental insecticide poisoning (Beyer et al., 1996). In C. batrachus' liver and muscle tissues, there have been similar increases observed in ALAT and AAT activities after a short exposure to dimethoate (Begum and

Vijayaraghavan, 1995, 1996). The increased activity of transaminases supplies the oxaloacetic acid, pyruvate, ?-ketoglutarate, and glutaric acid to cater to the enhanced energy requirements during conditions of stress induced by triazophos. These compounds, including oxaloacetic acid, pyruvate, and ?-ketoglutarate could have been piped into the citric acid cycle. The subsequent deamination of the resulting glutamic acid from tranamination could lead to the formation of ammonia (Bidigare and King, 1981), which is also supported by the increased ammonia levels that were observed.

There exists an enzyme system designed for swift mobilization of glycogen to initiate the release of energy. In this procedure, the crucial stage is forming glucose-1-phosphate from glycogen, facilitated by the effect of glycogen phosphorylase (Morgan and Parmeggiani, 1964).

Improvements in the functioning of GDH as a result of triazophos poisoning were noted in the liver and muscle tissues of C. punctatus. This escalation in GDH suggests a rise in glutamate oxidation, which subsequently leads to an increase in the production of ammonia and ?-ketoglutarate at the expense of NAD. Moreover, it appears that the production of ammonia based on glutamate is escalating, as indicated by the elevated GDH activity and higher levels of ammonia.

The current study's findings indicate that a disruption occurs in protein and carbohydrate metabolism in liver and muscle tissue when exposed to triazophos insecticide. Some parallels and contrasts were noticed between the organs, which typically align with their different functionalities, such as glycogen storage in the liver. These outcomes hint at the involved mechanisms and propose ways to identify them. This type of research on fish offers insights into the negative effects on aquatic life and the potential ecological consequences these

insecticides could cause in regions nearby agricultural fields.