The ameliorative effect of quercetin on bisphenol A-induced toxicity in mitochondria isolated from rats

Recent studies have demonstrated that bisphenol A (BPA) has an adverse or toxic effect on the kidney. This study was designed to evaluate the ability of quercetin (QUER) to prevent BPA-induced mitochondrial dysfunction. Thirty-two healthy adult male Wistar rats were randomly divided into four groups, as follows: control group (olive oil), BPA group (250 mg/kg), BPA þ QUER group (250 mg/kg + 75 mg/kg), and QUER group (75 mg/kg). All treatments were orally administered for 14 days. Kidney mitochondria were isolated by administration of the different centrifugation method. Uric acid and creatinine were considered to be biomarkers of nephrotoxicity. The ameliorative effects of QUER on BPA toxicity were evaluated by determining the glutathione (GSH) content, CAT, the damage to the mitochondrial membrane, the reactive oxygen species (ROS), and lipid peroxidation (LPO). Administration of BPA significantly decreased kidney weight. In the kidney, BPA can deplete GSH content and CAT activity, increase the mitochondrial ROS formation, and enhances LPO and mitochondrial membrane damage. The pretreatment of mitochondria with QUER has the ability to reduce the toxic effects of BPA in isolated mitochondria. These findings suggest a potential role for QUER in protecting mitochondria from oxidative damage in kidney tissue.

Keywords: Bisphenol A; Quercetin; Nephrotoxicity; Oxidative stress; Antioxidants; Biomarker

Bisphenol A (BPA) is a chemical compound that it has been reported to have widespread accumulation in human body. It is conjugated by glucuronic acid in liver and excreted in urine as BPA glucuronide and may also be considered a uremic toxin (Dekant and Volkel [19]). BPA exposure is determined as urinary BPA concentration because serum levels are very low when renal function is normal (Haishima et al. [32]). BPA is a xenoestrogen and its effect has been linked to the development of obesity, insulin resistance, and metabolic syndrome (Caporossi and Papaleo [14]; Jahan et al. [36]). BPA has structural similarity with phenols (Gonzalez-Parra et al. [30]), is a material of the plastics industry used in the production of diverse materials, and is the epoxy layer of canned food (Kubwabo et al. [43]; Rudel et al. [55]). Application of this chemical compound in industries is on the rise (Halden [33]). However, such widespread use raises concern that BPA could pose a risk to both ecosystems and human beings and BPA due to their small size (10 < φ < 100) can easily affect the body's organs and cause harm (Carwile et al. [15]; Vandenberg et al. [60]; Welshons et al. [62]). There have been reports on the effect of BPA in human tissues especially, the liver and kidney (Qin et al. [51]). The adverse effect of BPA on the kidney was demonstrated to include reactive oxygen species (ROS) generation with oxidative DNA and the alteration of kidney biochemical profiles as well as degeneration of renal tubules in kidney of rat and mice (Bindhumol et al. [11]). Kidneys have receptors for estrogen; BPA could stimulate the receptors, thereby leading to epithelial cell proliferation and also might increase the volume of proximal and distal tubule and cause hydronephrosis (Poormoosavi et al. [50]). The studies also report the involvement of oxidative stress and mitochondrial dysfunction to the damaging effect of BPA (Anjum et al. [6]; Chitra et al. [17]; Hassan et al. [34]; Mahmoudi et al. [48]). BPA induces oxidant stress within the renal parenchyma (Trasande et al. [59]) and is capable of acting directly on the kidney mitochondria, causing mitochondrial oxidative stress, dysfunction, and subsequently, leading to whole organ damage (Kim et al. [42]). Exposure to BPA results in the inhibition of the first complex of the electron transfer chain (Khan et al. [40]). Mitochondrial damage is caused by impairment of electron transport chain (Fig. 1) (Andreazza et al. [5]). Impairment of electron transport chain seriously damages the tissues and induction of oxidative stress (Nita and Grzybowski [49]). The studies also indicate relations between serum BPA levels and increased oxidative stress and inflammatory markers in hemodialysis patients (Bosch-Panadero et al. [13]). The studies also reported the beneficial effects of plant products (Ginseng, Aloe vera, Cordyceps militaris, and Grape seed extracts) as antioxidants against BPA toxicity (Behmanesh et al. [10]; Rameshrad et al. [52]; Saadeldin et al. [56]; Wang et al. [61]). Quercetin (QUER) (3,3′,4′,5,7-pentahydroxyflavone) is a plant pigment and an essential aglycone flavonoid that is present in cereals, vegetables, and fruits that are commonly eaten by human beings (Salvamani et al. [57]). It has many pharmacological applications, including anti-ischemic, antiallergenic, anti-cancerogenic, anti-inflammatory, and antiviral (Rice-Evans et al. [54]). Moreover, QUER protects the liver (El-Shafey et al. [22]) and kidney oxidative stress (Liu et al. [45]). Several epidemiologic studies have confirmed the association of QUER consumption with diseases that are significantly affected by inflammation and oxidative stress, such as cardiovascular disease, lung cancer, asthma, and other respiratory disorders (Gargouri et al. [27]; Huang et al. [35]). QUER is a potent antioxidant and can form a chelate with metal ions. Therefore, it is able to prevent the reaction Fenton (Cherrak et al. [16]). Numerous scientific studies also suggest that QUER has beneficial effects on kidney (Faddah et al. [24]; Khajevand-Khazaei et al. [39]; Lu et al. [46]).

Graph: Fig. 1Production of ROS in mitochondria

Sangai NP et al. showed that QUER decreased reactive oxygen species (ROS) production and increased the total antioxidant capacity in rat kidney (Sangai and Verma [58]). QUER caused the levels of these biomarkers to decrease, which indicates that QUER has beneficial effects on the kidney. In a study by Fartkhooni et al., the improvement of renal tissue was accompanied by a significant reduction in the plasma levels of these biomarkers (Fartkhooni et al. [26]). It seems that application of QUER can reduce the production of free radicals and play a significant role in the correction of antioxidant/oxidant balance. With this background in mind, this research aimed to evaluate the effect of QUER on PBA-induced mitochondrial toxicity in rat kidney.

All of the compounds used in the study, including D-mannitol, 2′,7′-dichlorofluorescin diacetate (DCF-DA), thiobarbituric acid (TBA), quercetin, reduced GSH, Coomassie blue G, 2-[4-(2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid (HEPES), tetraethoxypropane (TEP), sucrose, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), dithiobis-2-nitrobenzoic acid (DTNB), bovine serum albumin (BSA), ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), and rhodamine 123 (Rh 123), were provided by Sigma Chemical Co. (St. Louis, Missouri, USA).

The present study was conducted on 32 healthy adult male Wistar rats (8–10 weeks old and 180–200 g,) that were prepared from Ahvaz Jundishapur University of Medical Sciences. The animals were maintained in polypropylene cages at a controlled temperature (25 + 2 °C) and on a 12:12-h light-dark regimen, with free access to drinking water and standard rodent chow. This work was performed according to the guidelines of the Animal Ethics Committee of Ahvaz Jundishapur University of Medical Sciences (approval number: IR.AJUMS.REC. 1395.632).

The study was based on the block randomization method. The rats were randomly classified into four groups (N = 8) and were administered with one dose of BPA per day by intragastric intubation for 14 days. Based on the power calculation and estimation of attrition from our pilot study and previous studies, the sample size was determined to be 8 rats per group (Khorsandi et al. [41]). The control group (Group I) was given placebo (olive oil). Group II contained rats treated with olive oil + 250 mg/kg of BPA. The animals in group III received olive oil + 250 mg/kg BPA + QUER of a dose 75 mg/kg, and group IV were administered with olive oil + QUER of a dose 75 mg/kg. All treatments were orally administered for 14 days. The doses of BPA and QT were determined based on our pilot study and previous studies (Goloubkova et al. [29]; Khan et al. [40]; Zou et al. [64]).

Differential centrifugation of the rat kidney was carried out to prepare mitochondria. After the removal and mincing of the kidney by a small scissor, the kidney pieces were placed in a freshly prepared cold mannitol solution encompassing 10 mM HEPES-potassium hydroxide, 70 mM sucrose, 0.1% (w/v) BSA, 200 mM d-mannitol, and 1 mM EDTA at pH of 7.4. A glass homogenizer was applied to mildly homogenize the minced kidney, followed by the sedimentation of the broken cell debris and nuclei by centrifugation at 600×g and 4 °C for 10 min. Moreover, centrifugation of the supernatants was carried out in 10,000×g for 15 min. Afterwards, we carefully removed the upper layer and washed the pellet through mild suspension in the isolation buffer, followed by the centrifugation of the compound at 10,000×g for 10 min. Using the mannitol solution, the resulting mitochondrial pellets were suspended (Lawrence and Davies [44]). Moreover, the Biuret method was exploited to determine the concentrations of protein (Gornall et al. [31]).

We measured the mitochondrial ROS using the fluorescent probe DCF-DA. Then, incubation of the isolated kidney mitochondria (0.5 mg protein/ml) was carried out with 1.6 μM DCF-DA at 37 °C for 10 min. To measure the fluorescence at the emission and an excitation wavelength of 500 and 520 mm, respectively, we used the Perkin Elmer LS-50B Luminescence fluorescence spectrophotometer (California, USA) (Crow [18]).

We determined the MMP using the mitochondrial uptake of the cationic fluorescent dye (Rhodamine 123). The tubes were mildly shaken at 37 °C with 1.5 μM Rh 123 for 10 min for incubation of the mitochondrial kidney suspensions (0.5 mg protein/ml). Then, to estimate the fluorescence, the Elmer LS-50B Luminescence fluorescence spectrophotometer was applied at emission and excitation wavelength of 490 and 535 nm, respectively (Baracca et al. [9]).

For determine lipid peroxidation (MDA), the technique introduced by Zhang et al. was used. (Zhang et al. [63]). We applied 0.3 ml 0.2% TBA and 0.25 ml sulfuric acid (0.05 M) for incubation of mitochondrial suspensions (0.5 mg protein/ml). For 30 min after that, the tubes were maintained in a bath filled with boiling water. Finally, the tubes were transferred to an ice bath and butanol (0.4 ml) was poured into each tube. In the next stage, centrifugation of the tubes was carried out at 3500×g for 10 min. Following that, assessment of the total quantity of MDA shaped in each sample was carried out using a spectrophotometer (UV-1650 PC) to measure the supernatant's absorbance at 532 nm. Furthermore, MDA was expressed as nmol/mg protein and TEP was applied as the standard.

DTNB was applied by the spectrophotometric technique to determine the GSH contents in isolated kidney mitochondria (Ellman [23]). Then, 0.04% DTNB was mixed with the kidney mitochondrial suspensions (0.5 mg protein/ml) in 0.1 mol/l of phosphate buffers (pH 7.4). We read the development of the color yellow at 412 nm (spectrophotometer UV-1650 PC). The content of GSH was expressed as micrograms of protein per milligram of tissue.

The catalase activity was determined by the method of Claiborne's method (Armann et al. [7]). Initially, 50 mM of potassium phosphate (pH 7.0), 19 mM of H2O2, and 20 mL supernatant of homogenate mitochondria islets were mixed. The H2O2 was added to this mixture. The rate of H2O2 decomposition was assessed by measuring the absorbance changes at 240 nm for 60 s. One unit of CAT activity is defined as 1 mM of H2O2 that is consumed in 1 min. Ultimately, the specific activity of CAT was expressed as unit per milligram of protein.

In this part, we estimated some essential enzymes of the test of kidney function in the serum so that the BPA-induced toxicity of kidney could be evaluated. Blood samples were collected for biochemical analysis and centrifuged at 3000 rpm for 10 min to isolate the serum. The serum concentrations of uric acid, creatinine (Cr), and blood urea nitrogen (BUN) were determined spectrophotometrically using suitable kits.

Data are presented as means ± SEM. Comparisons of the differences were performed by a one-way analysis of variance (ANOVA) followed by Fisher's LSD for multiple comparisons or nonparametric Kruskal-Wallis as appropriate. Statistical significance was taken at P < 0.05. All analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, IL).

No significant differences in body weights were observed between control and other groups (P > 0.05). A significant reduction in kidney weights was observed in the BPA-intoxicated rats in comparison to the control (P < 0.001). Pretreatment of QUER could significantly increase the kidney weights compared to BPA-intoxicated animals (P < 0.001). These results are shown in Table 1.

Effects of quercetin on the kidney weight (g), initial and final body weights

Data expressed as mean ± SEM A significant difference in comparison to the control (***P < 0.001) Significant difference in comparison with the BPA-treated group (###P < 0.001)

As shown in Table 2, the creatinine and urea levels significantly increased in the BPA group compared with the control group (P < 0.001 and P < 0.01 respectively). In the groups that were administered with the QUER, the creatinine and urea levels decreased when compared with the BPA group (P < 0.01 and P < 0.05 respectively) (Table 2).

Effects of quercetin on the serum creatinine (μmol/l) and urea (mmol/l), kidney function tests in BPA-induced toxicity

Data expressed as mean ± SEM A significant difference in comparison to the control group (**P < 0.01; ***P < 0.001) A significant difference in comparison to BPA-treated group (#P < 0.05; ##P < 0.01)

As shown in Fig. 2, following BPA exposure (250 mg), ROS generation increased significantly in the isolated kidney mitochondria as compared to the control group (P < 0.001). However, ROS formation was significantly inhibited (P < 0.001) by QUER pretreatment.

Graph: Fig. 2ROS formation was measured fluorometrically using DCF-DA, as described in the "Materials and methods" section. A significant difference in comparison to the control (*** P < 0.001). A significant difference in comparison to BPA-treated group (### P < 0.001)

The alteration of MMP as an expression of the mitochondrial dysfunction induced by BPA was addressed. As shown in Fig. 3, BPA group rates revealed a substantial depolarization of ∆Ψm when compared to the control group (P < 0.001). However, the loss of ∆Ψm was partially reversed when co-treated with QUER (P < 0.001), although both values were still more than the control and QUER groups.

Graph: Fig. 3The effects of QUER on mitochondrial membrane damage in BPA-induced nephrotoxicity. A significant difference in comparison to the control (*** P < 0.001). A significant difference in comparison to BPA-treated group (### P < 0.001)

In the BPA-receiving group, compared to the control group, the glutathione level significantly decreased (P < 0.001). Administration of quercetin significantly increased the glutathione level, compared to the BPA group (P < 0.05) (Fig. 4).

Graph: Fig. 4Effects of pretreatment with QUER on the GSH levels in BPA-induced nephrotoxicity. A significant difference in comparison to the control (*** P < 0.001). A significant difference in comparison to BPA-treated group (# P < 0.05)

In the BPA group, compared to the control group, the MDA level significantly increased (P < 0.001). Administration of quercetin significantly reduced MDA level, compared to the BPA-receiving group (P < 0.01) (Fig. 5).

Graph: Fig. 5Effects of pretreatment with QUER on the MDA levels in BPA-induced nephrotoxicity. A significant difference in comparison to the control (*** P < 0.001). A significant difference in comparison to BPA-treated group (## P < 0.01)

Evaluation of the Fig. 6 showed that BPA significantly decreased the activity of catalase, compared to the control group (P < 0.001). Moreover, administration of 75 mg/kg of quercetin, compared to the BPA-receiving group, significantly increased the catalase activity level (P < 0.05) (Fig. 5).

Graph: Fig. 6Effects of pretreatment with QUER on the catalase activity in BPA-induced nephrotoxicity. A significant difference in comparison to the control (*** P < 0.001). A significant difference in comparison to BPA-treated group (# P < 0.05)

The major factors leading to mitochondrial injury are the perturbation of the mitochondrial respiratory chain and induction of oxidative stress (Khan et al. [40]). According to the previous studies, the lipophilic nature of BPA is responsible for the elevation of the oxidative stress level (Gassman [28]). Moreover, this compound elevates the formation of reactive oxygen species (ROS) and may activate the apoptosis, pathway of oxidative stress, and other cytotoxicity pathways (El-Beshbishy et al. [21]; Faheem and Lone [25]). There is an effective defense mechanism to neutralize the free radical-induced damage in body. This is proficient by endogenous antioxidant enzymes (such as CAT) and non-enzymatic antioxidant (such as GSH) (Ahangarpour et al. [3]). These enzymes constitute a mutually supportive team of defense against reactive oxygen species (ROS) (Birben et al. [12]). Lipid peroxidation and ROS generation were related to kidney toxicity induced by BPA and indirectly reflected a decrease in the antioxidant defense system. Thus, the attenuation of lipid peroxidation in BPA-treated rats by QUER provides a convincing evidence for the involvement of ROS in BPA-induced lipid peroxidation. Our findings showed that treatment of rats with 75 mg/kg QUER decreased BPA toxicity by decreasing the MMP. QUER reduces the levels of ROS, an oxidative stress marker, and MDA, the end-product of lipid peroxidation. Reduction in mitochondrial GSH levels has shown oxidative damage, because glutathione is an important biomarker of oxidative stress; it forms the first line of antioxidant defense against oxidant induced damage (Dutta et al. [20]); according to our observations, the GSH level significantly decreased in the kidney mitochondria of rats treated with BPA, compared to the control group; therefore, it could be indicated that rats treated with BPA aggravate the mitochondrial damage and viability through the oxidative stress and reduction of GSH level. The reduction of the level of GSH demonstrated its application in the detoxification of the free radicals (Aboul Ezz et al. [2]). MDA content is an index of the intensified peroxidation process (Ayala et al. [8]). In this study, MDA concentration in kidney tissue was significantly increased by BPA. QUER could attenuate the BPA-induced increase in the kidney MDA content. These effect reveal a state of oxidative stress in kidney mitochondria. It is obvious from the present data that high dose of BPA could induce oxidative stress in the kidney mitochondria. In this regard, our findings are in line with the results obtained by Kamel et al. studies, which indicated the increase of tissue MDA due to BPA exposure (Kamel et al. [38]).

The enzymatic antioxidant (CAT) activity was measured to estimate the stability of ROS production in the kidney mitochondria. The results of the present research were also indicative of a significant decrease in the level of activities of antioxidant enzyme CAT in kidney mitochondrial of the BPA group. In this study, the significantly reduced activities, CAT, indicated that BPA induced oxidative stress in renal tissue. However, pretreatment with QUER significantly reversed the activities of CAT, which indicated the anti-oxidant property of QUER.

When the kidney is damaged, serum Cr and BUN are released into the bloodstream and elevated concentrations of these indicate damage to the kidney cells (Alidadi et al. [4]). In this study, the increase in serum Cr and BUN concentration indicated that BPA toxicity can cause damage to kidney tissues. Thus, due to the anti-inflammatory and anti-oxidant properties of QUER, it may inhibit BPA-induced renal damage in rats.

The results showed that the weight of the kidneys decreased in the BPA group. In this study, QUER reversed the renal weight loss induced by BPA. This finding was in agreement with another study that reported that QUER mitigated the effects of bisphenol A on the body and organ weights of mice (Sangai and Verma [58]). The protection by QUER was observed in association with the restorations of oxidative parameters (GSH and MDA) in the kidney tissues, suggesting that QUER exerted its renoprotection via antioxidant mechanism that is compatible with publications showing the free radical scavenging and antioxidant properties of QUER (Abdel-Raheem et al. [1]; Renugadevi and Prabu [53]).

We examined the effect of BPA on mitochondrial dysfunction. Among several parameters, determination of MMP is one of the vital factors. The mitochondrial membrane potential (Δψm) is crucial for mitochondrial movement (Ly et al. [47]) and mitochondria with high Δψm were shown to preferentially move anterogradely (Kalmar et al. [37]). In the current research, BPA significantly induced MMP collapse (P < 0.001), which was reversed by QUER (Fig. 2). This indicates that BPA-induced MMP collapse is prevented by QUER (P < 0.001) (Fig. 2). In addition, QUER is the most effective agent in protection against mitochondrial dysfunction among different structurally related naturally occurring flavonoids (Kim et al. [42]).

The results of the isolated kidney mitochondria assessment in this research revealed that BPA could induce oxidative stress through the ROS overproduction, significant decrease in CAT and GSH activities, and enhanced lipid peroxidation in kidney. Moreover, mitochondrial damage has been occurred in BPA administration animals. The decreased activity of GSH during BPA toxicity might be due to the enhanced lipid peroxidation. The protective effects of QUER, as shown by its impact on GSH, CAT, and MDA, suggest that administration of antioxidants such as QUER may restore the mitochondrial antioxidant status, thus protecting these organelles from oxidant damage.

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