Dietary quercetin ameliorates TPT-induced hepatic oxidative damage and apoptosis in zebrash

The objective of this study was to determine the effects of quercetin on oxidative stress and apoptosis induced by TPT in zebrash. 240 sh were divided into 4 groups with three repeats. D1: sh fed with the basal diet as the control group. D2: sh fed with basal diet and exposed in 10 ng/L TPT. D3: sh fed diets containing 100 mg/Kg quercetin and exposed in 10ng/L TPT. D4: sh fed diets containing 100 mg/Kg quercetin. The results showed that quercetin could ameliorate oxidative stress, which decreased MDA, NO levels and improved antioxidant enzyme activities. The key apoptotic gene expressions, including caspase3, Bax and caspase9 mRNA expression were signicantly induced by TPT exposure as compared with the control group, while notably decreased the Bcl-2 gene. However, dietary quercetin prevented a signicant increase in Bax, caspase3 and caspase9 mRNA levels induced by TPT exposure, but increased Bcl-2 mRNA levels. The results of our study also demonstrated that 10 ng/L TPT signicantly up-regulated TNF-α, IL-1β, IL-8, and NF-kB p65 gene expression and down-regulated IL-10 and IkB expression compared to the control group. However, TPT-induced inammation was signicantly mitigated in the quercetin treatment group. In conclusion, our ndings suggested that quercetin might alleviate hepatic oxidative damage and apoptosis induced by TPT.

Although the use of TPT has been banned in many countries, there are still some reports of TPT contamination in coastal areas of China (Gao et al. 2017). It has reported that the seafood ( sh, sea vegetables, shell sh) contained TPT levels are higher than the TBT levels ). There has been a lot of concern about toxicological and eco-toxicological properties of organotin compounds (Clasen et al. 2017;Chen et al. 2019). An eco-toxicological evaluation of TPT has indicated that aquatic animals might face serious risks with long-term exposure to TPT (Wen et al. 2018). The aquatic products have posed the most exposure risk to humans. Thus, exploring the mechanism of TPT and protective measures is of great importance. In the present study, TPT was used to induce oxidative stress, and evaluated the antioxidant effect of quercetin. This information will be with great interest to explain a well understanding of the toxicology of TPT and the functional effects of quercetin.
Oxidative stress is due to the excessive production of free radicals and the decrease of the activity of the antioxidant defense system, which interferes the equilibrium state of the pro-oxidation system and the antioxidant system (Kennedy et al. 2005). The antioxidants play a pretty crucial role in the health of sh.
Excess reactive oxygen species (ROS) are toxic because they can attack and destroy a variety of biological molecules, such as DNA, carbohydrates, proteins and membrane lipids, leading to cell death and tissue damage. ROS is often used as a biomarker of oxidative stress, and its excessive production can induce oxidative damage and apoptosis (Patten et al. 2010). The in uence of ROS on the health of sh has become of increasing interest. Many studies have shown that TPT can induce oxidative stress in different aquatic organisms, such as copepods (Tigriopus japonicus) (Yi et al. 2014), green microalgae (Scenedesmus quadricauda) (Xu et al. 2011), rotifers (Brachionus koreanus) (Yi et al. 2016), male guppies (Poecilia reticulata) (Zhang et al. 2019) and abalones (Haliotis diversicolor) (Lu et al. 2016). TPT was found to inhibit the activities of the antioxidant enzymes such as glutathione S-transferases (GST), superoxide dismutase (SOD) and catalase (CAT) (Zhang et al. 2019). The cellular cytophysiological actions are regulated by the balance between the antioxidant capacity and the production of reactive oxygen species (ROS) (Parellada et al. 2012). Antioxidants such as quercetin, Vc, VE, can protect cells by scavenging free radicals. Antioxidant system includes different types of components, both enzymatic and non-enzymatic. The SOD, glutathione peroxidase (GPx) are both antioxidant enzymes, and other enzymatic antioxidants such as CAT can quickly remove the ROS, which can protect the body from their harmful effects (Li et al. 2013). However, how the TPT toxicity inhibits the antioxidants and the quercetin alleviate this response are not clear.
Apoptosis is the death process of cells in response to the physiological and pathological stimulus signal that plays a crucial role in the health of sh. It is an indispensable component of various cellular processes that mediate the phagocytosis to remove dying or infected cells, and remodel the amed sites by reducing the release of pro-in ammatory cytokines (Luo et al. 2017). Apoptosis is strictly regulated. Many factors including pro-apoptotic factors (Bax, Casp-3 and Casp-9), anti-apoptotic factors such as Bcl-2 could affect the process of apoptosis. So, these genes could serve as indicators to study the effect of TPT in zebra sh liver and the ameliorating effect of quercetin.
Quercetin is an important bioactive compound found abundantly in many vegetables, fruits, fruit juices and herbal dietary supplements (Wach et al. 2007). It is known to have anti-in ammatory, antioxidant, neuro-protection, anti-carcinogenic, antidiabetic, and anti-proliferative effects in vitro and in vivo (Meng et al. 2018;Dokumacioglu et al. 2018;Laura et al. 2018). In one study, quercetin was reported to scavenge ROS and can effectively inhibit neuronal damage by regulating the oxidative stress (Adedara et al. 2017).
In addition, quercetin is considered to be a natural avonoid for the prevention of hepatotoxicity (Oliveira et al. 2014). However, the molecular mechanisms of TPT-induced liver injury and protective effects of quercetin are not yet completely understood. The aim of this study was to ll these gaps. Thus, the antioxidant and anti-in ammatory capability of quercetin were evaluated in this study. In general, this research contributes to a better understanding of the regulatory of quercetin and its role in the management of liver injury.

TPT and quercetin
In this experience, TPT chloride (98%) was obtained from Sigma-Aldrich (USA). The concentrations of TPT was 10 µg Sn/mL (33.1 n M/mL), which was prepared in the 95% ethanol. At the time of exposure, the solutions were added directly to the water. Quercetin was also purchased from Sigma-Aldrich with a purity more than or equal to 95%.

Experimental diets
The proximate composition and ingredients of the basal diets are shown in Table 1. The dosage of quercetin was 100 mg/kg and then was mixed with the basal diet. The feed material were ground into ne powder with a shredder, and then mixed with appropriate water to form a soft dough. The dough was granulated (no steam injected) using a 0.3 mm diameter pellet grind. The experimental feed was dried overnight at 50℃ and stored in airtight bags at 4℃ until use.

Fish and experimental design
Zebra sh were provided from a commercial sh dealer (Guangzhou, China). Before the feeding trial, sh were acclimated for two weeks at the Aquarium Science Laboratory in Henan University of Science and Technology. During the period of acclimation, the sh were feeding a commercial diet with two times per day, which contained 11.32% moisture, 33.42% crude protein and 6.28% crude lipid. After the acclimation, the sh were starved for 48 h. Fish were randomly distributed into four groups, each with 60 sh. Every tank was placed 20 sh (mean weight of 2.05g ± 0.03g) in 60 L glass tanks. The tank contains 40 L dechlorinated tap water. The control group was fed the basal diet (D1) and the following three treatments were implemented. D2: sh fed with basal diet and exposed in 10 ng/L TPT. D3: sh fed with experimental diets and exposed in 10 ng/L TPT. D4: sh fed with experimental diets. In the process of experiment, sh were fed twice a day at 7:30, 18:30 respectively and water was exchanged by 25% daily at 8:30. The experiment lasted for 8 weeks. Water temperature was controlled at 26℃ to 27℃. pH :7.0 -7.5, The dissolved oxygen was maintained approximately at 6.0 mg/L.
Fish were dissected on ice. The livers were removed, frozen in liquid nitrogen and stored at -80°C for further enzymological assays and RNA quanti cation.

Assessment of oxidative stress
Frozen liver tissues were homogenized in 0.5 mL ice-cold 0.86% physiological saline with 10x volume (pH = 7.4) and centrifuged at 12,000 g for 10 min at 4°C. Then, supernatants were collected for various biochemical analyses. Oxidative stress induced by TPT was examined via measures of the MDA and NO levels. The MDA level was measured using the thiobarbituric acid. The quanti cation of NO release was detected by DAF FM DA uorescence assay as described Choi (Choi et al.2012). The glutathione (GSH) level was determined using the method of Sedlak and Lindsay with some modi cations (Sedlak and Lindsay 1968). The GSH levels were expressed as nmol/g tissue. The activity of SOD was measured using a SOD kit (Randox labs. Crumlin, UK), as described by Breinholt et.al (Breinholt et al. 2000). The activities of GPx and CAT were determined with the detection kits (Nanjing Jian-Cheng Bioengineering Institute, China) according to the manufacturer's protocol. Using bovine serum albumin as a standard, the total protein concentrations in the supernatants were determined by Bradford method (Bradford 1976).

Gene expression analysis
Total RNA was isolated from the hepatopancreas using TRIzol reagent (Takara), following the manufacturer's instructions. Using a Nanodrop 1000 spectrophotometer (Thermo Scienti c, USA) determined the RNA concentration by obtaining the UV absorbance at 260nm. The absorbance ratio at 260nm/280nm was determined to evaluate the RNA Purity. cDNA was synthesized from RNA with the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scienti c, USA). The reaction tube was placed on the PCR ampli cation instrument for 60min at 37℃ and 15min at 70℃ for denaturation. The reverse transcripts were stored at 4℃ for later use.
The TNF-α, IL-1β, IL-8, NF-kBp65, IL-10 and IkB, Casp-3, Casp-9, Bax and Bcl-2 expression were determined using Real-time PCR. PCR was performed using a 7300 Real-Time PCR System (Applied Biosystems) in 96-well plates (Axygen). The reaction system contained 20 µL mixtures using SYBR Green I dye. PCR ampli cations contained 1 µL cDNA template, 1 µL forward and reverse primers (concentration of 1pM), 6.8 µL of SYBR green PCR master mix and 10.2 µL nuclease-free water and. The PCR reaction tube was put into the DNA ampli cation instrument for 94 ℃ for 4 min, and then the thermal cycle was carried out under the following conditions: 95 ℃ for 45 s, 57 ℃ for 1 min, 72 ℃ for 1 min. The PCR was carried out for 40 cycles. Details of primers used in this study were shown in Table 2. Genes expression was expressed as multiple change relative to the control. They were calculated by 2 −ΔΔCt method with three replicates for treatment (Livak and Schmittgen). The expression pro les of target genes were normalized with β-actin (housekeeping or conserved gene).

Results
3.1. Effects of TPT and quercetin on hepatic oxidative damage parameters in the liver of zebra sh The effects of TPT and treatment with quercetin and their combination on lipid peroxidation and liver oxidative parameters are shown in Fig. 1. After exposure to TPT, the levels of MDA and NO were signi cantly increased, while GSH, GPX, SOD and CAT levels were signi cantly decreased compared with these indexes in control group (P < 0.05). However, the dietary quercetin signi cantly (P < 0.05) reduced the toxic effects of TPT on hepatic GSH, GPx, SOD, CAT, MDA and NO, and these values were higher or lower (P < 0.05) than the TPT group values. Interestingly, quercetin group showed signi cant elevation in the SOD and GSH levels compared with control group (P < 0.05).

Effects TPT and quercetin on apoptosis-related gene expression in the liver of zebra sh
The effects of quercetin on TPT-induced Bcl-2, Bax, caspase3 and caspase9 mRNA expression changes are shown in Fig. 3. Compared with the control treatment, TPT treatment signi cantly increased the mRNA expression of Bax, caspase3, and caspase9, while signi cantly decreased the expression level of Bcl-2 (P < 0.05). However, dietary quercetin prevented a marked increase in liver Bax, caspase3 and caspase9 mRNA expression induced by TPT exposure, but increased Bcl-2 mRNA levels (P < 0.05). 3.3. Effects TPT and quercetin on in ammatory response-related gene expression in the liver of zebra sh.
The effects of TPT and treatment with quercetin on in ammatory response-related gene expression are shown in Fig. 2. Compared with the control group, TPT signi cantly increased the levels of TNF-α, IL-8, IL-1β and NF-kBp65 mRNA expression levels of in zebra sh liver (P < 0.05). However, compared with the TPT group, dietary quercetin signi cantly decreased the mRNA expression levels of TNF-α, IL-8, IL-1β and NF-kBp65. The IL-10 and IkB mRNA expression were down-regulated by TPT exposure (P < 0.05). Quercetin decreased the changes in the expression of IL-10 and IkB decreased by TPT. In addition, administration of quercetin alone showed no signi cant difference (P > 0.05) compared to control groups (Fig. 3).

Discussion
Oxidative stress is unbalanced between the oxidation and antioxidation, which is considered an important role in many pathological and physiological phenomena of the body. Concerning oxidative stress indices, our study showed that sh exposure to TPT had signi cantly higher levels of the MDA and NO in liver tissue in zebra sh. On the other hand, MDA levels in liver tissues decreased after the application of 100 mg/kg quercetin compared to the TPT group. MDA is a scienti cally recognized indicator of oxidative stress, which is the product of polyunsaturated fatty acid peroxidation (Parvez and Raisuddin 2005). MDA levels rise when oxidative stress occurs (Del 2005). In general, the decrease of GSH level or GSH/GSSG ratio can express excess ROS (Jeroen et al. 2015). The liver synthesizes key enzymes that remove ROS and produce the systemic antioxidants, including GSH, SOD, GPx and CAT (Todorova et al 2005). Antioxidant enzymes are considered as the main defense system to inhibit the production of ROS and protect macromolecules from oxidative damage. SOD and GSH are the main antioxidant enzymes in the liver, and are also the main elements to enhance immunity to prevent diseases. While GPx is the main protective enzyme that can effectively scavenge free radicals in organisms, thus protecting cells from oxidative damage (Yildirim and Kilic 2011). In the present study, the antioxidant enzymes were examined after exposure to TPT. Based on the obtained results, it was shown that GSH level, GPx, SOD and CAT activities decreased in the TPT group. However, these enzyme activities in sh fed 100mg/kg quercetin were signi cantly increased in liver tissue. These results suggest that quercetin can protect liver against TPT-induced oxidative stress by regulating antioxidant enzyme activities. Quercetin effectively mitigates oxidative status by reducing lipid peroxidation and increasing various antioxidant enzymes in the liver of zebra sh. In previous study, quercetin could also scavenge oxidative radicals and increase the level of antioxidant defense (Livingstone 2001). In previous study, it was reported that quercetin increased the level of GSH, decreased the ROS and MDA levels, and regulated antioxidant enzyme activities in ethanol-exposure rats (Molina et al. 2003). Consistent with our experiment, a study showed that quercetin can elevate SOD and GPx activities and ameliorate MDA level (Adedara et al. 2017). Quercetin sweep free radicals off and thereby reduces oxidative and cytotoxic effects may be due to its molecular structure including several hydroxyls which can interact with free radicals to inhibit oxidative stress (Zhang et al. 2019). Whereas in another study, it was reported that quercetin attenuated the increases of antioxdant activities induced by dichlorvos (Salem et al. 2015). These different results might be related to animal species, breed conditions and animal state. This study demonstrated that dietary quercetin can effectively inhibit oxidative stress caused by free radicals.
The hepatotoxicity of TPT is closely related to the formation and apoptosis of ROS in hepatocytes. The production of oxidative stress and ROS are important signals of apoptosis (Todorova et al. 2005). The increased levels of ROS might impair the mitochondria functions, including changes of mitochondrial permeability, respiration and oxidative phosphorylation. Then, The mitochondria release cytochrome C, which activates caspase-3 and caspase-9, leading to cell apoptosis (Chen et al. 2014). It has also been reported that ROS mediate the mitochondria-dependent apoptosis in a variety of cells (Singh et al. 2007). Additionally, oxidative stress can damage DNA and directly or indirectly cause cell apoptosis (Tyor and Pahwa 2017). Bcl-2 is an anti-apoptotic gene and can inhibit cell apoptosis by preventing the release of cytochrome c from mitochondria. But Bax induce release of cytochrome c to trigger a pro-apoptotic pathway (Deng et al. 2009). If the Bax/Bcl2 ratio increases, it indicates the induction of cell apoptosis (Whiteman et al. 2007). In this study, it showed that several apoptotic related genes, such as Bax, caspase3 and caspase9, were up-regulated, while the mRNA expression of anti-apoptotic Bcl2 genes was decreased in the TPT group. We hypothesize that TPT lead to liver cell apoptosis by inducing of oxidative and activating p53 expression, which leads to the transcription of genes encoding pro-apoptotic proteins.
Cas3 (caspase-3) plays an irreplaceable role in cell apoptosis, which could be activated by Cas9 (caspase-9) and is a very important terminal splicing enzyme in the process of apoptosis (Deng et al. 2009). However, dietary quercetin attenuated cell apoptosis with downregulation of Bax, Casp-3 and Casp-9 as well as upregulation of Bcl-2. Quercetin has anti-apoptosis effect in various tissues (Kumar et al. 2014;Hu et al. 2015;Lei et al. 2015). Similar results were also found that 100 µ M of quercetin decreased apoptosis, while 1000 µ M of quercetin increased apoptosis (Ahn and Jeon 2015). We suggested that in the dietary quercetin group, the decrease of apoptosis might be related to the restrain of pro-in ammatory cytokines in liver. However, the underlying mechanism in sh remains unclear, which require further research.
Numerous researches have shown that organotin caused in ammation and pathological alterations Zhang et al. 2017;Zhang et al. 2018). TNF-α, IL-1β and IL-8 are major proin ammatory cytokines and, they are often used as markers of the in ammatory response (Secombes 2016). But the anti-in ammatory cytokines, such as IL-10 and TGF, may reduce the overreaction on in ammatory response in sh (Kemenade et al. 2009). Paralleled with the oxidative stress parameters, the TNF-α, IL-8 and IL-1β mRNA levels were signi cantly increased compared to the control. But, dietary quercetin remarkably alleviated the in ammatory response induced by TPT, showing lower gene expressions of pro-in ammatory cytokines, indicating that quercetin might alleviate the in ammation in the liver of sh. Although several previous studies indicated that quercetin attenuated animal in ammation mediated by inhibiting the expression of pro-in ammation gene (Julie et al. 2012;Mehta et al 2017). No studies of the possible effects of quercetin on sh have been reported. The quercetin regulated the pro-in ammatory cytokines, which might be related to the related signaling such as NF-kB. NF-kB plays an important role in the regulation of pro-in ammatory cytokines such as IL-8 and TNF-a (Neurath and Pettersson 1997). In this study, compared with the TPT group, quercetin decreased the mRNA expression of NF-kB p65 in the liver of zebra sh. Further contrastive analysis showed that the TNF-a, IL-1β and IL-8 were positively correlated with the mRNA expression of NF-kB p65, which suggested that quercetin inhibited the levels of TNF-a, IL-1β and IL-8 might be related to the decrease of NF-kB gene expression. In addition, NF-kB inhibitor protein (IkB) can directly inhibit NF-kB in sh (Iwasaki et al. 2011). In mice, it has been reported that the up-regulation of NF-kB mRNA was regulated by the downregulation of IkB gene expression (Beg et al. 1995). In this study, the author rstly showed that TPT downregulated IkB mRNA levels in the liver of zebra sh, where quercetin up-regulated this gene. Further analysis indicated that the IL-8, TNF-a mRNA levels and NF-kB p65 were negatively correlated with the IkB mRNA levels, suggesting that quercetin might be through up-regulation the IkB mRNA level and downregulation the NF-kB mRNA level to prevent in ammatory response in sh. Similar results were also observed in mice (Jung et al 2012).
This result indicated that TPT exposure could induce accumulation of MDA and NO in tissue, inhibiting the antioxidant system. Quercetin could protect the zebra sh liver against TPT-induced injury by decreasing ROS production, alleviating cell apoptosis, renewing antioxidant enzymes activities, and attenuating in ammation.    caspase9) in the liver of zebra sh. Data were expressed as mean ± SEM and analyzed by One-way ANOVA, followed by turkey's multiple range test (n=9). Different letters denote signi cant difference (P < 0.05).