Protective Effects of Tea Leaf Extract (Camellia sinensis) Against Cypermethrin-Induced Toxicity in Gill and Liver of Rainbow Trout (Oncorhynchus mykiss)

Pesticides have widely been used to increase agricultural productivity and to control undesired pests and diseases for centuries (Karatas et al., 2019a). Although there are many legal regulations regarding pesticides, it has caused an important rise in pesticide use worldwide because of its wide spectrum and low cost (Pearson et al., 2016; Ben Slima et al., 2017). Pesticides contain highly toxic substances, and their direct or indirect contamination into water resources through agricultural, industrial, and domestic runoff poses a major risk to aquatic organisms including fish (Weston et al., 2005; Struger and Fletcher, 2007; Shelley et al., 2009). For this reason, fish are considered one of the most important organisms in toxicity studies. In recent years, it has been determined that pyrethroid pesticides are more toxic and used more than organophosphates due to their high sensitivity in living things (Karatas et al., 2019a). One of the widely used pesticides among pyrenoids is cypermethrin (CMN). CMN is one of the photostable synthetic pyrethroids and is the active ingredient of commonly used insecticides (Stephenson, 1982). CMN is highly toxic to fish, and the 96 h LC50 value for rainbow trout is between 0.5-8.2 µg/L (Stephenson, 1982, 1983; Bradbury and Coast, 1989). The daily Kow for CMN is 6.06 and its solubility in water is 0.01 mg/L and the half-life in water is more than 50 days (Struger and Fletcher, 2007; Shelley et al., 2009).

The tea plant belongs to the genus Camellia of the Theaceae family. Tea leaves (Camellia sinensis) contain flavan-3-ols (catechins), flavonol glycosides and phenolic acids (Clifford et al., 2000; Yao et al., 2004; Qin et al., 2022). About 75% of the polyphenols in the tea leaf are flavanols, and 60-70% of the flavanols are epigallocatechin-3-gallate (Katiyar and Mukhtar, 1997). Aslo, the flavanols are known to scavenge free radicals, have powerful antioxidant properties, inhibit hydrolytic and oxidative enzymes (phospholipase A2, cytochrome oxygenase, lipoxygenase), and anti-inflammatory activities (Kinsella et al., 1993; Zhishen et al., 1999; Oda and El-Maddawy, 2012). However, there is no study on the protective efficacy of tea leaf extract (TLE) on rainbow trout. This study was carried out to determine the protective efficiency of TLE on blood biochemistry (total protein, albumin, AST and ALT), oxidative stress, immunity, antioxidant enzymes, histopathology, apoptosis, and DNA damage against the toxicity induced by CMN in gill and liver tissues of rainbow trout (Oncorhynchus mykiss).

After purchasing healthy rainbow trout, they were kept in the stock pond for 15 days for adaptation. During this time, the fish were fed commercial trout feed (Abalıoğlu, Turkey), (Karatas et al., 2020). After the adaptation period, 10 fish for each tank were stocked in tanks with a flow of 0.5 L and a water volume of 380 L (temperature 11.3°C; dissolved oxygen 7.95; and pH 6.8) (Karatas et al., 2019a).

Fresh tea leaves were gathered from Rize, Turkey, dried in the room temperature, and pulverized using a grinder. Then, the mixture containing ethanol (300 ml) and sample (100 g) was prepared. This mixture was kept in 96% ethanol solution for 24 h at room temperature and filtered. Ethanol was removed under vacuum. Then, 50 and 100mg doses of the prepared TLE were added to commercial trout feed (Mohamed et al., 2018; Karatas et al., 2020).

Rainbow trout with weight of 40 g at the beginning of the experiment were randomly divided into 4 groups, each of 10 fish. Until the end of the trial, two groups (group 1 and 2) were fed with commercial trout feed, group 3 fed with 50 mg TLE and group 4 was fed with 100 mg dose of TLE added to commercial trout feed. No deaths were observed in the groups throughout the trial. Then, groups 2, 3 and 4 were exposed to 10 % of the LC50 value of CMN (CAS Number 52315-07-8, ≥98%, Molecular Weight 416.30) obtained from Sigma–Aldrich (Germany) for 21 days.

Blood from the fish tail vein was drawn into vacuum-sealed gel serum tubes, where it was allowed to coagulate for seven minutes. Then, the coagulated blood was centrifuged at 3000 rpm for 10 min. and separated into serum (Karatas et al., 2021). Serum metabolites such as total protein, albumin, aspartate aminotransferase (AST), serum alanine aminotransferase (ALT) (Kit no: 3183734190) were measured by a Cobas 6000 autoanalyzer (Karatas et al., 2019a, 2020). White blood cell (WBC) was measured using the sysmex XN9500 modular system. T. Ig level in fish was determined as described by Siwicki and Anderson (1993).

After removing the gill and liver tissues, they dissected using Tissue Lyser II. The tissues were diluted with 1.15% potassium chloride to obtain 1:10 (w/v) homogenate for MDA, GSH and protein analysis. The homogenates were centrifuged at 3500 rpm for 15 min for MDA and protein analysis, and at 11000 rpm for 20 min for glutathione (GSH) analysis (Karatas et al., 2021). Protein concentration in supernatants (liver and gill tissues) was measured spectrophotometrically at 650 nm according to the Lowry method using standard bovine serum albumin (Lowry et al., 1951). MDA was determined at 532 nm as described by Placer et al. (1966). GSH was determined at 412 nm as described by Stahr (1977). The superoxide dismutase (SOD) activities were determined at 560 nm as described by Sun et al. (1988). The catalase (CAT) activities was determined at 405 nm as described by Goth (1991).

After the gill and liver tissues were removed, they were kept in 10% formalin for 48 h. Tissues taken from formalin were cut in 4 μm thickness and embedded in paraffin blocks. Then, sections were stained with Hematoxylin-Eosin (HE) and observed under microscope (Bar: 50µm) (Karatas et al., 2019b, 2021).

The histological sections passed through xylol and alcohol series for immunoperoxidase analysis were kept in 3% H2O2 for 10 min. for endogenous peroxidase inactivation after washing with PBS. To determine the antigen in the tissues, they were treated with antigen retrieval solution for 2x5 min at 500 watts in the oven. As shown in the immunohistochemistry kit process (AbcamHRP/ DAB Detection IHC kit), tissues were incubated with 8-OHdG and Caspase 3 Antibody (Catalog no: sc66036, sc-56053, dilution 1/50; Santa Cruz, USA) for 30 min at 37˚C for detection of DNA and apoptotic cell damage. The chromogen was 3-3’ Diaminobenzidine. The sections were showed according to their immune positivity (Karatas et al., 2019a).

SPSS 20.0 program was used for all of the statistical analyses of this study. ANOVA (one way analysis of variance) test was used to determine the statistical differences of the results obtained from the study, Duncan test was used in multiple comparisons and p<0.05 was considered significant. Kruskal-Wallis (nonparametric) test was used for semi-objective findings in histopathology and Mann Whitney U test was used for pairwise comparisons.

The changes in liver functions of the groups exposed to CMN toxicity after being fed with 50 and 100 mg doses of tea leaf extract are given in Table I. Serum total protein, and albumin levels were decreased in the group 2 exposed to CMN (p <0.05). In contrast, total protein and albumin levels in groups 3 and 4 were increased compared to group 2 (p <0.05). ALT and AST activities, known as liver enzymes, were increased in the group 2 (p <0.05). However, these enzymes were decreased in groups 3 and 4 (p <0.05). There was no statistically significant difference between the liver functions of groups 3 and 4 (Table I).

CMN exposure led to a significant increase in MDA levels and a significant decrease in SOD, CAT activities and GSH levels in the liver and gill tissues of group 2 compared to group 1 (p<0.05). However, while MDA levels

The results were given as mean and standard deviation. Different letters indicate differences between groups. Group 1: Control; Group 2: CMN exposure; Group 3: CMN exposure after feeding with 50 mg of tea leaf extract; Group 4: CMN exposure after feeding with 100 mg of tea leaf extract.

ALT, alanine aminotransferase; AST, aspartate aminotransferase.

The results were given as mean and standard deviation. Different letters indicate differences between groups. For details of groups, see Table I.

CAT, catalase; GSH, glutathione; MDA, malondialdehyde; SOD, superoxide dismutase.

The results were given as mean and standard deviation. Different letters indicate differences between groups. For details of groups, see Table I.

T. Ig, total immunoglobulin; WBC, white blood cells

decreased in the liver and gill tissues of the groups 3 and 4, SOD, CAT activities and GSH levels increased (p<0.05). Moreover, group 4 was more effective in reducing the toxicity effects caused by CMN compared to group 3 (Table II).

T. Ig and WBC were reduced in the group 2 exposed to CMN (p <0.05). In contrast, T. Ig and WBC levels in groups 3 and 4 were higher than those of group 2 treated with CMN (p <0.05). There was no statistically significant difference between the liver functions of groups 3 and 4 (Table III).

None (−), Mild (+), Moderate (++), and Severe (+++). For details of groups, see Table I.

The liver and gill tissues of the groups 1 had normal histological appearance (Figs. 1A, 2A). CMN exposure caused severe degeneration, steatosis, inflammation, necrosis, hyperemia, enlargement of sinusoids and moderate mononuclear cell infiltration in the liver tissue (Fig. 1B), and severe degeneration, desquamation, necrosis of the lamellar epithelium, as well as severe adhesion in the lamella due to cell infiltration in the interlamellar spaces and dilatation in the vessels in the gill tissue of group 2 (Fig. 2B). While 50 mg dose of TLE caused moderate degeneration, moderate steatosis, mild inflammation and mild necrosis in the liver hepatocytes (Fig. 1C) and severe degeneration, severe desquamation, moderate adhesion, and mild necrosis in the gill lamellar epithelium of group 3 (Fig. 2C), 100 mg dose TLE caused mild degeneration and steatosis in liver hepatocytes (Fig. 2D), and mild adhesion and proliferation in gill interlamellar epithelium of group 4 (Fig. 2D) (Table IV).

8-OHdG and caspase 3 expressions in the liver (Figs. 3A, 5A) and gill (Figs. 4A, 6A) tissues of groups 1 were negative. Expressions of 8-OHdG and Caspase 3 of group 2 were severe in liver hepatocytes (intracytoplasmic localization) (Figs. 3B, 5B) and gill lamellar epithelium (Figs. 4B, 6B). The 8-OHdG and Caspase 3 expressions were moderate in liver hepatocytes (intracytoplasmic) (Figs. 3C, 5C) and gill lamellar epithelium (Figs. 4C, 6C) of group 3 and mild in liver (Figs. 3D-5-D) and gill (Figs. 4D, 6D) tissues of group 4 (Table V).

None (−), Mild (+), Moderate (++), and Severe (+++). For details of groups, see Table I.

In recent years, studies have focused on the safety, dosage and action mechanisms of different plants in the protection of living things against pesticides (Rahmani et al., 2015). One of these plants is tea. There are many different types of tea, such as green, black and white, which is an important nutritional component. Tea, which contains active phytochemicals (polyphenols), has an active role in protection against pesticides and diseases due to its rich antioxidant source (Khan and Mukhtar, 2007). The main polyphenol of tea is epigallocatechin-3-gallate (EGCG; 59%) and it has therapeutic effects in protection against pesticides and diseases by regulating biochemical, physiological and molecular processes (Singh et al., 2010).

AST and ALT have high sensitivity to hepatotoxicity and histopathological changes, including amino acid metabolism (Stoyanova et al., 2016), and are used to evaluate the level of liver tissue damage such as cell inflammation and necrosis caused by pesticides (Ullah et al., 2014; Karatas et al., 2019a, b). CMN exposure led to increase in serum ALT and AST activities in group 2. The increase in these enzymes may be the most important cause of liver dysfunction and damage. However, there were significant decreases in serum AST and ALT activities in groups 3 and 4. This may be an indication of the protective effect of tea leaf extract on the liver. Our results were similar to those of studies on animals such as the African catfish (Sayed and Soliman, 2018) and Wistar rat (Ahmed et al., 2019). Moreover, there are significant decreases in serum protein and albumin levels of group 2 exposed to CMN. These reductions may be related to decreased oxygen availability, inhibit energy production, suppress oxidative metabolism, as well as hypoxia due to lactic acid accumulation (Karatas et al., 2019a). Similar results were observed in common carp exposed to bifenthrin (Velisek et al., 2008), brown trout exposed to deltamethrin (Karatas et al., 2019a) and rainbow trout exposed to tetrachlorobiphenyl and diazinon (Vijayan et al., 1997; Banaee et al., 2011; Karatas et al., 2019b).

Reactive oxygen species (ROS) could lead to the cellular and molecular changes such as lipid, protein, DNA and antioxidant, damage the membranes by making them permeable, and different physiological instabilities lead to necrosis and thus apoptosis (Livingstone, 2001; Lushchak, 2011; Topal et al., 2017; Ullah et al., 2019). Reduced SOD, CAT and GSH and increased MDA in liver and gill tissue of group 2 may be due to excessive free radical production (Saxena et al., 2011). Histopathological results support these findings. However, the reduce in MDA levels and increment in CAT and GSH levels in groups 3 and 4 may be a result of suppressing the formation of reactive oxygen molecules and free radicals, as well as preventing oxidative stress. Moreover, the increase in SOD activities may be associated with the detoxification of superoxide radicals (O2-) (Ullah et al., 2019). The increase in both of these antioxidants, even under stress, may be an indication that tea leaf extract supports the defense system. Similar results have been observed in African catfish exposed to 4-NP (Sayed and Soliman, 2018) and winstar rat exposed to doxorubicin (Ahmed et al., 2019). Moreover, there was a significant decrease in T. Ig and WBC levels of group 2 treated with CMN compared to group 1. Decreased WBC and T. Ig may be the result of chronic and/or persistent immunosuppression of prolonged CMN exposure (Ullah et al., 2018). Moreover, the reduction in protein content may be partially associated with the decrease in WBC levels which are the main source of protein production such as immunoglobulin, lysozyme, complementary factors, and bactericidal peptides (Misra et al., 2006a, b; Soltanian and Fereidouni, 2017). However, there was an increase in T. Ig and WBC levels of groups 3 and 4. Hasanpour et al. (2017) reported that green tea supports the immune system such as T. Ig, lysozyme, and ACH50. Our results confirm that the natural antioxidants in tea leaf extract support the immune system against CMN toxicity and overcome the formation of necrosis in liver and gill tissues. Histopathologically, severe degeneration, desquamation, necrosis and adhesion in the gill epithelium and severe steatosis, degeneration, necrosis, inflammation and hyperemia in the liver tissue of group 2 exposed to CMN were observed. One of the deadly effects of pyrethroids such as cypermethrin and deltamethrin on fish is gill damage. Therefore, pyrethroids have a high absorption rate by gill even at low levels due to their high lipophilicity (Smith and Stratton, 1986; Velisek et al., 2006; Ullah et al., 2019; Karatas et al., 2019a). The liver is considered to be responsible for detoxification of toxic substances in living things. Karatas et al. (2019a) showed that hyperemia may be a result of increased inflammation in liver tissue. Colakoglu and Donmez (2012) determined that impaired lipid biosynthesis or lipid transport is the most important cause of steatosis and degeneration in the liver. Salim et al. (2011) and Karatas et al. (2019a) showed that toxic substances such as aflatoxins and deltamethrin cause hepatocyte necrosis in sinusoids due to the expansion and swelling of hepatocytes. Different researchers determined different lesions in fish such as Rohu exposed to CMN (Sarkar et al., 2005), Rainbow trout exposed to CMN (Velisek et al., 2006), Common carp exposed to CMN (Dobsíková et al., 2006), Clarias gariepinus exposed to CMN (Velmurugan et al., 2009), Zebrafish exposed to CMN (Paravani et al., 2018), and Nile tilapia exposed to CMN (Korkmaz et al., 2009). However, 100 mg dose TLE against CMN toxicity prevented necrosis, hyperemia and inflammation in liver tissue as well as necrosis in gill tissue and significantly reduced degeneration and steatosis in the liver and desquamation and adhesion in the gill tissue of group 4. Sayed and Soliman (2018) determined that green tea extract reduced the hepatotoxic effects of 4-NP in catfish.

8-OHdG is one of the markers used to determine the levels of DNA damage caused by ROS (Cadet et al., 2003; Stepniak and Karbownik-Lewinska, 2016; Gelen et al., 2021). Hydroxyl radicals formed due to oxidative stress are an indicator of hydrogenation of nucleic acid leading to 8-OHdG (Cadet, 2016; Gelen et al., 2021). The 8-OHdG expression level was severe in both liver and gill tissues of the group 2 exposed to CMN. The increase in the 8-OHdG expression levels in liver and gill tissues may be due to oxidative stress and superoxide anion (O2) production (Onouchi et al., 2012; Anjana et al., 2013; Karatas et al., 2019a, b). Previous studies showed that pyrethroids increase 8-OHdG expression levels (Arslan et al., 2017; Karatas et al., 2019a, b). The severe of 8-OHdG expression caused by CMN in liver and gill tissue was significantly decreased in the groups 3 and 4. This suggests that the powerful antioxidant agents found in tea leaf play an important role in reducing DNA damage caused by oxidative stress.

Apoptosis has a vital effect on the survival of multicellular organisms by getting rid of damaged or infected cells that cannot perform their normal functions (Portt et al.,2011; El-Bakry et al., 2017; Teles et al., 2019). Caspase 3 is an effector protein that plays an important role in the mitochondrial and the death receptor pathways (Lavrik, 2010; Arslan et al., 2017). Caspase-3 expression levels were severe in gill and liver cells of group 2 exposed to CMN. It shows that increased apoptosis in liver and gill tissues may be associated with oxidative stress. Topal et al. (2014) reported that chlorpyrifos increases gill and liver cell apoptosis in rainbow trout and can activate caspase-3. The severity of caspase 3 in groups 3 and 4 was lower than that in group 2. Reduced apoptosis in liver and gill tissues may be the result of suppressing oxidative stress and increasing antioxidant defense system of flavonoids found in tea extract (Ahmed et al., 2019). In addition, Spencer et al. (2001) determined that 3′-O-methyl epicatechin found in tea reduces caspase-3 activity by inhibiting H2O2-induced cell death.

Consequently, TLE had positive effects on all processes such as suppression of oxidative stress, strengthening of immune and antioxidant defense system, regulation of the effects of inflammatory signaling pathways and anti-apoptotic. Especially, 100 mg dose of tea leaf extract was determined to be more effective in preventing liver and gill tissue damage caused by CMN. Further studies are needed to determine the effects of TLE on different tissues against different pesticides in rainbow trout.

The authors thank Ataturk University, Faculty of Aquaculture, Inland Water Fish Research and Application Center. A part of this work was presented at the 2lst National Fisheries Symposium.

This work was supported by the Scientific Research Project Coordination Unit of Agri Ibrahim Cecen University (Project number: SHMYO.20.001).

The experimental protocol was approved by ethics committee of Agri Ibrahim Cecen University.

This study was performed within the ethical rules determined by Agri Ibrahim Cecen University (Writing and decision number: 47825/207).

The authors have declared no conflict of interest.