Assessment of 8-hydroxy-2-deoxyguanosine activity, apoptosis, acetylcholinesterase and antioxidant enzyme activity in Capoeta umbla brain exposed to chlorpyrifos

Capoeta umbla individuals (weight 92.7–121.4 g; length 18–23 cm) caught in the Murat River (Bingöl, Turkey; 38°46'24.0"N; 40°35'40.7"E) were used in this study. The fish were transferred to 600 l fiberglass tanks at Bingöl University, Faculty of Agriculture, Aquaculture Facility. Prior to the experiment, the fish were acclimatized (21 days) following the fluid analysis procedure. During acclimation, they were fed a commercial diet ad libitum. The physicochemical properties of the water used in the study are presented in Table 1.

The insecticide used in this study was obtained from a commercial institution (Turkey). The CPF concentrations of 200, 300, 400, 500 and 1000 μg/l were used to determine the LC₅₀ value of CPF. Ten fish were randomly selected for each group from 600 l tanks and five groups were formed. The fish were placed in 60 l aquariums with aeration stones placed there beforehand. The effects of CPF were observed by the researchers for 96 h and changes were noted. Dead fish were immediately removed from the aquarium. Probit analysis was performed to calculate the LC₅₀ value after the 96-hour period.

After calculating the 96 h LC₅₀ value, C. umbla specimens (n = 180) were assigned to three groups (n = 30 per aquarium). The experimental groups were established in duplicate. The stock solution of CPF was prepared by dissolving in water. The following groups were established: I.

Control Group

Fish were exposed to the test solutions for 96 h. Fish samples were collected at 24 and 96 hours in accordance with the test procedure for the established application volumes. No fish deaths were observed at this stage of the experiment.

A commercial off-the-shelf Fish ELISA kit (Catalog No. 201-00-0041; SunRed) was used to determine the level of 8-OHdG in brain tissue homogenates of C. umbla fish. Homogenates were incubated for 10 min in the dark at 37°C on a plate shaker. Stop Solution (50 μl) was then added and the measurement was performed in an ELISA plate reader at an absorbance of 450 nm within 10 min (Alak et al. 2021).

A commercially available off-the-shelf fish ELISA Kit (CASP3; Catalog No. 201-00-0031; SunRed) was used to determine caspase-3 enzyme activity in control and experimental group samples. The main point of this analysis is to determine the product formed as a result of the reaction of the substrate with the caspase-3 enzyme. Measurements were performed in the ELISA plate reader at an absorbance of 450 nm within 10 min (Alak et al. 2021).

The AChE activity was determined by the modified method of Ellman et al. (1961). Brain tissues were homogenized in 0.1 M phosphate buffer (1% v/v, pH 7.4) containing Triton-X 100 and centrifuged at 13,000 x g for 30 min (4ºC). The obtained supernatants were used as a source of enzymes for the determination of AChE activity (Rosenfeld et al. 2001; Alak et al. 2019b).

Protein concentration was determined spectrophotometrically at 595 nm according to the Bradford method with bovine serum albumin as a standard (Bradford 1976).

One-way ANOVA was used for data analysis, and the significant difference between the control and experimental groups was determined with the Duncan test and SPSS 23.0 software. The required level for statistical significance was set as p < 0.05. The mean ± standard error (SE) form was used to represent the data (n = 15).

DNA is the most important biological target for oxidative attacks, and the 8-OHdG level is a commonly used marker for the evaluation of the genotoxic effect of pollutants (Alak et al. 2019b,c; Uçar et al. 2020b). Several published studies have reported that simulative pesticide toxicity triggers oxidative stress with ROS production at the 8-OHdG level and causes DNA damage (Alak et al. 2017a; Alak et al. 2019a, b; Uçar et al. 2020a; Atamanalp et al. 2021). This is because the effect of pesticides directly as an oxidizing agent can be induced by a wide variety of DNA lesions in the presence of cellular reductants (Song et al. 2009). In our study, CPF significantly induced 8-OHdG activity in the brain tissue of fish in parallel with the exposure time, which had a significant effect on 8-OHdG activity. In different studies conducted in parallel with our research, it has been reported that different pesticides increase the level of 8-OHdG in fish brain tissue (Topal et al. 2015; Topal et al. 2017a; Alak et al. 2021). Shi et al. (2010) stated that chemicals cause apoptosis and are associated with DNA fragmentation and caspase-3 activation.

Caspase-3, a member of the cysteine-dependent aspartate-specific protease family, is a central apoptosis agent that contributes to the morphology of apoptotic cells and endamages several molecules (Monteiro et al. 2009). Caspase-3 is an important parameter in the progression of apoptosis, which catalyzes the cleavage of certain essential cellular proteins (Teng et al. 2019). Alak et al. (2019a) reported that caspase-3 activation causes DNA fragmentation and cell death in the final application of apoptosis. In this study, however, it has been determined that the caspase-3 activation level in C. umbla brain tissue increased as a function of dose and time (96 h, 55 and 110 μg l^-1). The source of this increase may be the induction of apoptosis due to CPF toxicity. The determined increase in the caspase-3 level at a high dose and long exposure time is supported by parameters such as a decrease in GR, GPx, SOD and CAT enzyme activity, high MDA levels, and an increase in 8-OHdG levels. Caspases, as effectors of apoptosis, play an important role in many physiological processes such as maintenance of homeostasis, development of tissues and organs, maturation of cytokines, and immune responses (Qu et al. 2018). Apoptosis has a vital effect on the development and homeostasis of organisms. High levels of oxidative stress caused by ROS generation stimulate a variety of cellular signaling pathways, often associated with apoptosis. Stimulation of apoptosis by environmental toxicants is associated with a change leading to oxidative stress in the redox balance of the antioxidant defense system. It can be said that this change causes oxidative stress, DNA damage and apoptosis in fish. In addition, it can be considered that this increase occurs in apoptotic cells due to the deterioration of the body enzyme balance and the alteration of the apoptosis pathway (Alak et al. 2020; Uçar et al. 2020b).

AChE is one of the enzymes responsible for cholinesterase activity in nervous systems of vertebrates, and when exposed to pollutants, AChE can be used as a biomarker for the assessment of neurotoxic changes (Gholami-Seyedkolaei et al. 2013; Topal et al. 2017b). AChE is an important enzyme involved in the breakdown of the neurotransmitter acetylcholine into choline and acetate and may be a target for toxic chemicals (Schmidel et al. 2014). These chemicals, by inhibiting the AChE enzyme, lead to impaired nerve function and excessive ACh accumulation (Bhattacharya 1993). AChE inhibition occurring in the brain causes changes in behavior (Kirby et al. 2000). In this study, while no significant change was found in fish brain tissue exposed to 55 μg l^-1 CPF for 24 h, a significant decrease in AChE enzyme activity was determined in fish brain tissue exposed to 110 μg l^-1 for 24 h and 55 and 110 μg l^-1 for 96 h. When AChE activity decreases, acetylcholine accumulates in the synapses and this causes physiological deterioration in many functions such as feeding, swimming and behavior (Glusczak et al. 2006). The occurrence of AChE inhibition may be associated with lower AChE expression (Xing et al. 2013) and cause neurotoxic changes in the nervous system (Da Cuna et al. 2011). Furthermore, CPF affects this enzyme inhibition by disrupting the antioxidant defense system and inducing oxidative stress (Adedara et al. 2018). Previous studies have shown that pesticides can cause AChE inhibition in different fish tissues (Almeida et al. 2010; Xing et al. 2010; Xing et al. 2013; Topal et al. 2017a, b; Parlak 2018; Alak et al. 2019a).

Endogenous antioxidants such as GR, GPx, SOD and CAT, which are the first step of the defense mechanism, can effectively remove the produced free radicals (Bhattacharjee & Sil 2006) and protect cells against oxidative damage. Reduced CAT activity changes the redox state of the cell (Stara et al. 2012). The CAT enzyme is widely distributed in biological tissues and breaks down hydrogen peroxide into oxygen and water to protect tissues against oxidative damage (Vasylkiv et al. 2011; Parlak 2018). GPx catalyzes the reduction of hydrogen peroxide and lipid peroxides and acts as an effective protective enzyme against lipid peroxidation for the sake of using GSH (İspir et al. 2017). GR activity serves to maintain the cytosolic concentration of reduced glutathione. Stimulation of GR activity is a potential biochemical marker of oxidative stress (Cazenave et al. 2006; Kirici et al. 2021). If reduced GR and GPx activity is not compensated for by the synthesis of new glutathione molecules, it can lead to GSH depletion (Stara et al. 2012; Kirici et al. 2021). GSH is the substrate of GR and GPx enzymes. Reduction in GSH levels results in increased free radical production and disruption at the cellular level. GSH is important in the assessment of oxidative stress and inhibits hydrogen peroxide (Jurma et al. 1997). Changes in the GSH level under the influence of pollutants are explained by adjustments and activations that can replace the GSH level (Zhang et al. 2005). In this study, the decrease in GR and GPx activity in the CPF treated groups is believed to be associated with an increase in O₂ (Alak et al. 2017a, b). It can be said that the decrease in antioxidant enzyme activity as a result of CPF application suppresses the adaptation response of fish to this pesticide and cannot neutralize the ROS produced as a result of pollutants. Furthermore, the decrease in antioxidant levels can be attributed to the increase in free radicals by CPF and the excessive use of antioxidants to maintain this balance.

The SOD-CAT system acts as the first line of defense against oxygen toxicity due to its role in preventing oxidant formation. Therefore, changes in SOD and CAT activity are used as biomarkers to indicate ROS production (Tabassum et al. 2015). SOD is responsible for the removal of hydrogen peroxides, which is considered an important enzyme in the formation of lipo-peroxides and in the prevention of cell membrane damage (Oliva et al. 2010). In this study, reduction in GR, SOD, CAT and GPx activity in fish brain tissue showed that CPF exposure interrupted the defense line against toxicity and inhibited enzyme activity in tissues. Therefore, the increase in the lipid peroxidation level in target tissues is due to the accumulation of OH. This can be confirmed by the sustained increase in MDA levels in our study. MDA, one of the end products of lipid peroxidation that occurs under the influence of toxic substances, is an important indicator showing that the damaged structure of cell membranes is stable (Toroser et al. 2007). The decrease in antioxidant parameters sensitized cells to the adverse effects of oxidative stress and consequently increased lipid peroxidation supported the MDA production (Fırat & Aytekin 2018). Studies have shown that oxidative stress caused by synthetic pyrethroids leads to different acute toxicities and weakening of the immune system of fish at the later stages (Ullah et al. 2018; Uçar et al. 2020b; Atamanalp et al. 2021).

In this study, the LC₅₀ value of CPF was determined for the first time for C. umbla fish. Furthermore, based on the results, it was determined that CPF causes neurotoxic changes in fish brains by affecting physiological and biochemical functions. It was shown that CPF can be neurotoxic to fish through inhibition of antioxidant enzymes, increase in the MDA level, inhibition of AChE enzyme, increase in 8-OHdG and caspase-3 activity. These data may provide useful information in understanding the molecular mechanism involved in CPF toxicity and in understanding future ecotoxicological studies. In addition, the possible relationship between oxidative stress and biochemical-physiological response should be investigated in further studies with different aquatic organisms using long exposure time.