2023 Volume 2 Issue 1

Effect of Vanillic Acid and Morin on Bisphenol S and Diethyl Phthalate Induce-Nephrotoxicity in Male Rats


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Abstract

Endocrine disruptor chemicals (EDCs) have an external influence that has a detrimental effect on the biological system. Morin and vanillic acid has key nephron-pharmacological effects to reduce this effect. We used a rat model to study the impact of Morin and vanillic acid on diethyl phthalate (DEP) and bisphenol S (BPS)- induced nephrotoxicity. After twenty-one days of DEP (50 mg/kg) and BPS (200 mg/kg) exposure and treatment with Morin and vanillic acid (25 and 25 mg/kg), samples were taken for the evaluation of biochemical parameters, including glutathione peroxidase (GPx), glutathione (GSH), catalase activity (CAT), and superoxide dismutase (SOD). Levels of calcium, sodium, urea, and creatinine. Nitric oxide (NO), hydrogen peroxide H202, and malondialdehyde levels (MDA) respectively. The kidney membrane was considerably (P< 0.05) preserved by morin and vanillic acid therapy. In a strikingly significant way (P <0.05), co-treatment with Morin and vanillic acid reversed DEP+BPS-induced reductions in glutathione levels, CAT, SOD, GPx, and GSH activities, as well as calcium, sodium, urea, and creatinine levels in the kidney, while attenuating DEP+BPS-mediated increases in nephron-oxidative damage markers (MDA, H202, and NO levels). By acting as an antioxidant, scavenger of free radicals, and having nephron-pharmacological effects, morin and vanillic acid in combination at the same acute dosages may prevent DEP and BPS-mediated nephrotoxicity dysfunctions.


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Vancouver
Eteng OE, Bassey N, Eteng EI, Okwe EP, Ekpo G, Ekam V, et al. Effect of Vanillic Acid and Morin on Bisphenol S and Diethyl Phthalate Induce-Nephrotoxicity in Male Rats. Bull. pioneer. res. med. clin. sci.. 2023;2(1):25-34. https://doi.org/10.51847/JipHmYy6fi
APA
Eteng, O. E., Bassey, N., Eteng, E. I., Okwe, E. P., Ekpo, G., Ekam, V., & Ubana, E. (2023). Effect of Vanillic Acid and Morin on Bisphenol S and Diethyl Phthalate Induce-Nephrotoxicity in Male Rats. Bulletin of Pioneering Researches of Medical and Clinical Science, 2(1), 25-34. https://doi.org/10.51847/JipHmYy6fi

Effect of Vanillic Acid and Morin on Bisphenol S and Diethyl Phthalate Induce-Nephrotoxicity in Male Rats

Ofem Effiom Eteng1*, Nseobong Bassey2, Eteng Ibiang Eteng3, Ekam Patrick Okwe3, Grace Ekpo2, Victor Ekam2, Eyong Ubana2

1Department of Biochemistry, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria.

2Department of Biochemistry, University of Calabar, Cross River State, Nigeria.

3Collage of Nursing and Midwifery Science Itigidi, Cross River State, Nigeria.  


Abstract

Endocrine disruptor chemicals (EDCs) have an external influence that has a detrimental effect on the biological system. Morin and vanillic acid has key nephron-pharmacological effects to reduce this effect. We used a rat model to study the impact of Morin and vanillic acid on diethyl phthalate (DEP) and bisphenol S (BPS)- induced nephrotoxicity. After twenty-one days of DEP (50 mg/kg) and BPS (200 mg/kg) exposure and treatment with Morin and vanillic acid (25 and 25 mg/kg), samples were taken for the evaluation of biochemical parameters, including glutathione peroxidase (GPx), glutathione (GSH), catalase activity (CAT), and superoxide dismutase (SOD). Levels of calcium, sodium, urea, and creatinine. Nitric oxide (NO), hydrogen peroxide H202, and malondialdehyde levels (MDA) respectively. The kidney membrane was considerably (P< 0.05) preserved by morin and vanillic acid therapy. In a strikingly significant way (P <0.05), co-treatment with Morin and vanillic acid reversed DEP+BPS-induced reductions in glutathione levels, CAT, SOD, GPx, and GSH activities, as well as calcium, sodium, urea, and creatinine levels in the kidney, while attenuating DEP+BPS-mediated increases in nephron-oxidative damage markers (MDA, H202, and NO levels). By acting as an antioxidant, scavenger of free radicals, and having nephron-pharmacological effects, morin and vanillic acid in combination at the same acute dosages may prevent DEP and BPS-mediated nephrotoxicity dysfunctions‎.

Keywords: Oxidative stress, Nephrotoxicity, Morin, Vanillic acid, Diethyl phthalate, Bisphenol S


Introduction

Morin bio-flavonoids have a range of medicinal properties, such as anti-inflammatory, anti-hepatotoxic, and anti-ulcer actions. Despite the many different bioflavonoids, Morin was one of them to garner widespread notice in nature. Morin has a variety of pharmacological effects, including the ability to scavenge free radicals, the ability to inhibit xanthine oxidase, anti-inflammatory properties [1], the ability to protect DNA from free radical damage, and anticancer properties. Both traditional herbal remedies and diets contain morin hydrate [2].

Morin is thought to be a potential medicinal drug for a variety of ailments, most of which are brought on by free radical damage [3]. Reduced oxidative stress, decreased expression of tumor markers, and prevention of tumor development have all been seen after morin treatment.

Toxicants exposure to biological system poses or triggered free radicals generation. From a chemical standpoint oxidative stress is linked to either a rise in the generation of oxidizing species or a marked decline in the efficiency of antioxidant defenses like glutathione. The effects of oxidative stress depend on how significant these changes are; a cell can overcome minor disruptions and return to its initial form. However, more extreme oxidative stress may result in necrosis, whereas stronger stressors may result in necrosis. Even mild oxidation can produce apoptosis [4].

Because its administration has been shown to increase the bioavailability of chemotherapeutic medications by blocking P-glycoproteins, morin serves a protective effect in lowering carcinogenic improvement [5]. Morin promotes the production of anti-oxidant proteins such as Catalase, Superoxide dismutase, and Glutathione peroxidase, according to several investigations. Numerous studies have demonstrated that phytochemical substances can protect cell viability by lowering the ROS burden in cells. Studies have shown that flavonoids can block transcription factors or regulatory enzymes that are crucial for regulating inflammatory mediators. Strong antioxidants with the capacity to lessen tissue damage are known as flavonoids.

Using a natural phytochemical substance is a growing method for treating, delaying, or curing illness. The bioactive components from Morin are being used in this most recent study on nephrotoxicity therapy. To demonstrate the impacts of the phytochemicals, the potential and constraints associated with pharmaceutical development are also investigated. This study field examines possible druggability in addition to scientific soundness [6].

The controlled activities of Morin, which function through regulating numerous cell-signaling pathways, are the mechanisms of such inflammatory and anti-oxidant qualities [7].

As a natural phenolic acid component and a benzoic acid derivative, vanillic acid (VA) is utilized in food companies as a flavor, preservative, and additive. It is a kind of vanillin oxide that is created when ferulic acid is transformed from vanillin. The pharmacological effects of VA include an anti-metastatic action [8], anti-melanogenesis [9], antioxidant, anti-angiogenesis [10], and anti-apoptotic effects [11]. Recent research has demonstrated how VA can improve cardiac dysfunction and reduce oxidative stress after ischemia-reperfusion injury [12]. It has been discovered that diethyl phthalate (DEP) has a variety of acute and long-term harmful iinfluences on various species at many trophic places, as well as endocrine-disrupting qualities [13]. DEP may also be thought of as an oily, flavorless material that is utilized to enhance the functionality and longevity of several items [14]. To keep plastic polymers flexible, it is added as a plasticizer. It has been utilized in several goods, such as toys, automobile parts, tool handles, tape, rubber, toothbrushes, and plastic films. BPS is a synthetic organic molecule that is frequently emloyd as a antecedent in materials including epoxy resins and other types of polycarbonate plastic. The industry has adopted bisphenol S (BPS) as a secure substitute for BPA, however, new research has revealed a correlation between various BPS concentrations and oxidative stress [15]. It exists in the environment and can be emitted into the air directly from manufacturing facilities or consumer goods. Due to the widespread use of bisphenols in food and beverage containers, environmental pollution, skin contact with bisphenol-containing products, and food contamination are the main causes of global human exposure [16]. The structure of bodily organs may be significantly altered by chronic exposure to DEP and BPS have lipophilic qualities that allow them to pass through kidney membranes and start the process of creating free radicals that can harm cells [17].

Free radicals, ions, or molecules produced from molecular oxygen are known as ROS. They have a strong tendency to react negatively with biological components such as lipids, proteins, and DNA. Therefore, the purpose of this study was to clarify the mechanisms by which vanillic acid and Morin work in concert to counteract the harmful effects of DEP and BPS-induced nephrotoxicity in the rat model.

Materials and Methods

Chemicals and reagents

Vanillic acid (Cat. No. V 1240), morin (Cat. No. M 2357), and bisphenol S (Cat. No. D 5095) are three examples.  Dimethylsulfoxide (DMSO) was acquired from Libertas Laboratory Services Limited, Abeokuta. Diethyl phthalate DEP (Cat. No: D 1785) was purchased from Otto Chemie Pvt Ltd (Mumbai, India). Except as otherwise noted, all other compounds were made by British Drugs House Compounds Limited in Poole, Dorset, England.

Animal care

Male albino experimental animals Wistar rats were inbred in the Animal House, Department of Biochemistry, Federal University of Agriculture, Abeokuta, Nigeria. The rats ranged in weight from 150 g to 200 g. They were kept inside a suspended plastic cage in temperature-controlled (25 °C) rat housing with a typical 12-hour light/12-hour dark cycle. The rats were given regular pellet food and free access to fresh water after a week of acclimatization. According to the guidelines in the "Guide for the Care and Use of Laboratory Animals" created by the National Academy of Science (NAS) and distributed by the National Institutes of Health, all the animals were treated humanely. With approval number BCH/20160914, the organization gave the go-ahead for this trial.

 Experimental protocol

A straightforward randomization procedure was used to allocate 25 rats into 5 groups (n = 5) and treat them as shown in Table 1. Based on the 3R (replacement, reduction, and refinement) principles, five rats per group were employed [18]. BPS and DEP were mixed and administered at toxicologically relevant doses of 200 and 50 g/kg body weight (b.wt.) based on earlier investigations by [19] and [20]. Vanillic acid and morin were given immediately after the injection of BPS+DEP at doses of 25 and 25 mg/kg b.wt. based on a prior work that shown that morin has antioxidant properties at these dosages [21]. All treatments were administered using dimethyl sulfoxide (DMSO), and the duration was twenty-one (21) days.

 

Table 1. Grouping of experimental animals and their treatments.

GROUPS (n = 5)

TREATMENT

A

DMSO (0.4% v/v)

B

DEP (50 mg/kg b.wt.) + BPS (200mg /kg b.wt.)

C

BPS + 50 mg/kg b.w + DEP (200 mg/kg b.wt. Morin 25 mg/kg b.w+ Vanillic acid 25 mg/kg b.w)

D

Morin 25 mg/kg b.w + Vanillic acid 25 mg/kg b.w

Preparation of serum

The animals were slaughtered by anesthesia using Ketamine/Xylazine (100 and 7 mg/kg, respectively) [22] after the experiment, 24 hours after the previous treatment, and were dissected. Plain tubes were used to collect kidney samples. For 10 minutes, the samples were centrifuged at 5000 rpm. The clear supernatant known as the serum was taken out and utilized for the biochemical assessment.

In an erythrocyte lysate that had been properly diluted, antioxidant levels were found.

Antioxidants and oxidative stress markers

Post-nuclear supernatants from the homogenized (10% w/v) kidney organs were utilized in the following tests using a buffer (pH 7.4).

Catalase, excess hydrogen peroxide (H2O2) was reacted with ammonium molybdate to create catalase (CAT) activity, which resulted in a yellow complex that could be measured at 405 nm [23].

According to Glutathione Peroxidase (GPx) test, excess glutathione reacts with DTNB to generate a compound that absorbs at 412 nm [24].

 The amount of reduced glutathione (GSH) was measured using the procedure described in [25].

Observing the suppression of pyrogallol auto-oxidation at 420 nm served as the basis for the superoxide dismutase (SOD) experiment, which was previously described [26].

Nitric oxide (NO) and H2O2 production were quantified using the techniques described in [27] and [28], respectively.

According to the technique of [29], the oxidative breakdown of lipids and proteins was measured as malondialdehyde (MDA), and MDA was identified as thiobarbituric acid reactive substances (TBARS).

Using Bethlot Searcy's approach [30], the levels of calcium, sodium, urea, and creatinine were measured to assess nephrotoxicity. According to the directions on the diagnostic kits that were acquired from Randox labs (UK), the levels of sodium ions (Na+) and calcium ions (Ca+) were measured using a direct spectrophotometric technique to determine [31].

Histopathological examination

For histological investigation, kidney samples were maintained in a 10% neutral formalin solution. eosin with hematoxylin (H&E) were used to stain 5-mm slices of liver tissues that had been fixed in paraffin wax.

Statistical analysis

The data were shown as the mean and SEM for each group. The homogeneity of the groups was assessed using Analysis of Variance (ANOVA). If there was heterogeneity, the groups were separated using the Duncan Multiple Range Test (DMRT). A 0.05 p-value was deemed statistically significant. All statistics were performed using SPSS (Statistical Package for Social Sciences) software for Windows version 20 (SPSS Inc., Chicago, Illinois, USA). The graphs were made using Graph Pad Prism 8 Software (Graph Pad Software Inc., San Diego, USA).

Results and Discussion

Figure 1 depicts how exposure to diethyl phthalate and bisphenol S affected the levels of glutathione (GSH) in the kidney. When compared to the control group, the kidney GSH concentration in the DEP+BPS-exposed group decreased significantly (p< 0.05). As opposed to the DEP + BPS group, exposed groups treated with 25 and 25 mg/kg of Morin and vanillic acid, respectively, had a substantial rise (p< 0.05) in kidney GSH concentration. In addition, the kidney GSH concentration was significantly lowered (p < 0.05) in the group receiving Morin and vanillic acid treatment compared to the control group.

 

a)

b)

c)

Figure 1. Effect of Morin pretreatment on diethyl phthalate and bisphenol s mediated decrease in antioxidants in rats in the kidney. (a) The activities of glutathione s-transferase (b) the activities of glutathione peroxidase (c) the activities of catalase. Bars represent mean ±SEM (n=5). Bars with different letters are significantly different at P< 0.05.

 

GPX activity in the kidney was decreased after exposure to DEP and BPS compared to control. However, after treatment with 25 25 mg/kg of Morin and vanillic acid, respectively, there was a substantial increase in kidney glutathione peroxidase (GPX) activity (p< 0.05) in comparison to the DEP+BPS. Furthermore, as compared to the control group, the group that received Morin and vanillic acid had a significantly higher level of kidney glutathione peroxidase (GPX) activity (p <0.05).

Rats exposed to DEP and BEP exhibited kidney SOD activity levels that were considerably lower than the control group (p 0.05). However, there was a significantly increased level of kidney SOD activity in the groups treated with 25 and 25 mg/kg of vanillic acid and morin, respectively, compared to the DEP + BPS group (p 0.05). There was no difference between the group that had Morin and vanillic acid treatment and the control group (p > 0.05).

Similar to this, the kidney CAT was considerably lower in the DEP + BPS-exposed group in comparison to control group (p <0.05). When compared to the DEP + BPS group, the exposed animals managed with 25 and 25 mg/kg of Morin and vanillic acid, correspondingly, had significantly higher kidney CAT activity (p <0.05). However, as in comparison to the control group, there was no discernible difference between the Morin and vanillic acid-treated groups.

The impact of Morin and vanillic acid on indicators of oxidative damage in the kidney of DEP + BPS-exposed rats is shown in Figure 2. When compared to the control group, the kidney MDA levels in the DEP + BPS-exposed group increased significantly (p <0.05). The kidney MDA concentration, on the other hand, was significantly reduced under treatment with Morin (25, 25mg/kg), and this effect was dose-dependent. In addition, there was no discernible difference between the Morin and vanillic acid-treated group and the control group.

 

a)

b)

c)

Figure 2. Effects of Morin pretreatment on diethyl phthalate and bisphenol s mediated increase in oxidative stress markers in rats on kidney parameters. (a)  MDA level (b) Nitric oxide (c) Hydrogen peroxide. Bars represent mean ±SEM (=5). Bars with different letters are significantly different at P<0.05.

Similar trends were seen between the effects of therapy with vanillic acid and morin and exposure to DEP + BPS on renal NO levels. Although there was a dose-dependent reduction in reactive nitrogen oxide (i.e., NO) following treatment with Morin and vanillic acid, the kidney NO concentration in the exposed group was considerably lower (p <0.05) than that in the control group. The group that just had Morin therapy, however, did not significantly differ from the control group (p > 0.05).

The effects of exposure to DEP+BPS on kidney H202 indicate a similar pattern of substantial increase (P<0.05) exposed group compared to the control group. Hydrogen peroxide is triggered treatment with Morin and vanillic acid. When compared to the control group, there was a dose-dependent reduction after treatment with Morin and vanillic acid.

Kidney creatinine, urea, calcium, and sodium levels as a result of exposure to DEP+BPS while comparing to the healthy control group, all electrolyte values are significantly higher (P< 0.05) in Figure 3. When comparing to the control group, the management usuimg 25 mg/kg of Morin and 25 mg/kg of vanillic acid causes a significant drop in renal electrolytes.

 

a)

b)

c)

d)

Figure 3. Effect of Morin pretreatment on diethyl phthalate and bisphenol s mediated decrease in kidney function test (a) calcium (b) sodium (c) urea (d) creatinine. Bar represents mean ± SEM (n=5). Bar with different letters is significantly different at P < 0.05

The outcomes of the histopathological investigation are shown in Figure 4. The segment of kidney tissues from group A (0.4% DMSO) showed that no deaths were seen across all groups throughout this trial. The animals in the control group's kidneys were histologically examined, and the cortical and modular tubules were found to be normal without any evidence of tissue abnormalities, such as crystallization.