# Roles ofMoringa oleifera Leaf Extract in Improving the Impact of High Dietary Intake of Monosodium Glutamate-Induced Liver Toxicity, Oxidative Stress, Genotoxicity, DNA Damage, and PCNA Alterations in Male Rats

Authors: Tarfa Albrahim; Manal Abdulaziz Binobead
Journal: Oxidative Medicine and Cellular Longevity
 (2018)

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## Abstract

It is common for food to be made more palatable through the use of the flavour enhancer monosodium glutamate, also known as vetsin powder. The purpose of the study described in this paper was to explore how vetsin-induced hepatic toxicity, DNA fragmentation, damage, and oxidative stress modifications could be mitigated with moringa leaf extract (MLE). To that end, 40 male rats were separated into four groups: normal control, positive control or MLE, vetsin, and vetsin combined with MLE. Results indicated that, compared to the control group, the levels of serum alanine aminotransferase (ALT), aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), liver malondialdehyde (MDA), DNA damage, injury, PCNA, and P53 expressions were significantly enhanced by the administration of vetsin (P<0.05). However, the vetsin group had significantly reduced levels of albumin, globulin, total protein, liver glutathione (GSH), superoxide dismutase enzyme (SOD), catalase, and glutathione S-transferase (GST) enzyme activities (P<0.05) by comparison to control. Meanwhile, modifications in liver functions, oxidative stress, DNA damage, liver injury, and PCNA expression were alleviated when vetsin was administered alongside MLE. The authors conclude that vetsin may have many side effects and that MLE can ameliorate biochemical changes, oxidative stress, hepatic injury, PCNA, and P53 alterations induced by vetsin administration.


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## Body

## 1. Introduction

Vetsin (monosodium glutamate) is derived from amino acids, or glutamate, and is a popular flavour enhancer used to generate a savoury or umami taste in a variety of foods worldwide. Vetsin is commonly found in Asian cuisine and thus often associated with Chinese restaurants, but it is now frequently found in the Western diet, too [1].From a structural perspective, there is no difference between the glutamate molecules disseminated from the gastrointestinal tract to the circulation, irrespective of the type of food from which they derive [2]. The distinctive flavour it imparts makes numerous foods, often prepared on an industrial scale, appealing to consumers; consequently, food or food ingredients with its umami taste and flavour enhancement receive a great deal of attention in the food industry. Flavouring agents can promote consumption of foods that naturally lack flavour; thus, they are of nutritional significance [1]. Vetsin (monosodium glutamate) has been proven toxic for both humans and experimental animals [3]. For example, El-Atrash et al. [4] reported that monosodium glutamate induced liver toxicity and oxidative stress in rats.The edible treeMoringa oleifera can be found in the Asian and African tropical and subtropical regions and is valuable not only nutritionally but also pharmacologically [5–7]. Traditional medicine throughout the world has extensively employed various parts of moringa, including leaves, roots, bark, fruit flowers, immature pods, and seeds, to prevent and treat a wide range of afflictions, such as inflammation, infectious diseases, and cardiovascular, gastrointestinal, haematological, and hepatorenal disorders [7–9]. Moringa leaves contain natural antioxidants, comprising a number of phytochemicals of medicinal importance, such as the tumour-targeting glucosinolates and isothiocyanates [5, 9–12]. The ability of moringa leaf extract (MLE) to alleviate vetsin-triggered liver toxicity, apoptosis, damage, and oxidative stress changes therefore constituted the focus of our study.


## 2. Materials and Method

### 2.1. Chemicals and Drug

One kilogram of monosodium glutamate (vetsin) powder was obtained from Qingdao Huifenghe MSG Co. Ltd., China. For the MLE, freshMoringa oleifera leaves were purchased between May and June from farmers in Riyadh. The leaves were air-dried in a laboratory for five days and ground into fine particles using a simple hammer mill. The MLE was prepared by mixing one gram dried and powdered leaves with 10 ml boiling water for five minutes, according to Berkovich et al. [13].


### 2.2. Experiment Set-up and Rat Categorisation

Forty male albino rats were sourced from the animal house of the College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia, each with a weight of between 110 and 130 g and aged 9-10 weeks. The rats were kept in cages in suitable environmental conditions (22–24°C, 12-hour light/dark cycle) and were put on a diet of commercial pellet, without water restrictions. All of the experiments were conducted in compliance with the guiding principles for the care and use of laboratory animals of King Saud University in Riyadh, Saudi Arabia. The experiments were initiated 14 days after the animals were procured to allow them to become accustomed to the laboratory setting.The male rats were separated into four groups with ten rats in each, and over the four weeks of the experiments, all rats were given the same amount of food. The first group was the control group, which did not undergo any treatment. The second group was subjected to intragastrical administration of 200 mg/kg body weight of MLE over the course of four weeks. The third group was subjected to intragastrical administration of 5 mg/kg body weight vetsin powder over the course of four weeks. The fourth group was subjected to intragastrical administration of 5 mg/kg body weight vetsin powder combined with 200 mg/kg body weight MLE over the course of four weeks.When the experimental work was completed, the rats were subjected to overnight fasting. For the purposes of clinical chemistry, the rats were euthanized at the end of the fourteenth week, to permit the sampling of blood via retro-orbital puncture with the use of blood capillary tubes without heparin, in keeping with specifications. Following 10-minute incubation at room temperature, the blood samples were allowed to coagulate before being subjected to 15-minute centrifugation at 3000 rpm. After collection, the serum was isolated and stored in clean stopper plastic vials at −80°C in preparation for the examination of the serum parameters.All experimental procedures were performed in accordance with King Saud University’s ethical standards for experiments involving animals.


### 2.3. Determination of Serum Enzymes

The approach proposed by Schumann and Klauke [14] was adopted to analyse the activities of serum aspartate transaminase (AST) and alanine transaminase (ALT) with the use of a commercial kit (Human, Germany). The same kit was employed to analyse the activity of serum alkaline phosphatase (ALP) in keeping with the approach of Moss and Henderson [15]. The approach proposed by Doumas et al. [16] was followed to measure the levels of serum albumin and globulin with a commercial kit (Diamond, Egypt). Furthermore, the approach suggested by Szasz and Persijn [17] was used for approximation of GGT activity.


### 2.4. Preparation of Tissue

At the end of the experimental period, rats from each group were euthanized and subjected to a complete necropsy after 10–12 hr of fasting. Liver tissues were weighed, cut, and homogenized (10% w/v) separately in ice-cold 1.15% KCl 0.01 mol/l sodium potassium phosphate buffer (pH 7.4) in a Potter-Elvehjem type homogenizer. The homogenate was centrifuged at 10,000 g for 20 min at 4°C and the resultant supernatant was used for different enzyme assays.


### 2.5. Determination of the Biomarkers of Liver Oxidative Stress

The approach proposed by Mesbah et al. [18] was adopted to identify the composition of hepatic malondialdehyde (MDA). The approaches proposed by Aebi [19], Misra and Fridovich [20], and Habig et al. [21] were, respectively, used to identify catalase (CAT; EC 1.11.1.6) enzyme activity, superoxide dismutase (SOD; EC 1.15.1.1) enzyme activity, and glutathione S-transferase (GST; EC 2.5.1.18) enzyme activity in hepatic homogenate. Furthermore, the approach proposed by Ellman [22] helped to determine reduced glutathione (GSH) content in hepatic homogenate.


### 2.6. Analysis of Genotoxicity and DNA Damage in Hepatic Tissue

DNA damage analysis and quantification were conducted with the comet assay (single cell gel electrophoresis) method. 100 mg samples of liver were obtained from every experimental group and subjected to mincing in a chilled homogenising buffer consisting of 0.075 M NaCl and 0.024 M Na2EDTA, before undergoing gentle homogenisation with the use of a homogenizer (Ikemoto Scientific Technology Company Ltd., Japan) in ice. A suspension of cells was subjected to a 10-minute 700× g centrifugation at 4°C, followed by resuspension in cold buffer. An epifluorescence microscope permitted visualisation of the slides. The DNA migration length (comet tail length) caused by genotoxicity was established by scanning digital images in an image analyser in the case of the control and MSG groups. Observation of at least 505 arbitrarily chosen cells per sample allowed the determination of tail length (TL), representing the distance between the comet head and the final signal in the tail that could be seen, the DNA tail intensity (TI), representing the DNA fraction in the tail divided by the DNA quantity in the nucleus multiplied by 100, and the DNA tail moment (TM), representing the product of the DNA quantity in the tail and TL. Every assay was carried out three times and contrasted with control.


### 2.7. Histopathological Examination

Liver tissue samples were fixed with 10% formalin solution for 48 hours and then processed for paraffin sectioning. Sections were stained with Ehrlich’s haematoxylin and counterstaining with eosin was applied for histopathological examination according to Bancroft and Stevens [23].


### 2.8. Proliferating Cell Nuclear Antigen (PCNA) Expression

As proposed by Tousson et al. [24], an Avidin–Biotin–Peroxidase immunohistochemical method (Elite–ABC, Vector Laboratories, CA, USA) was applied to analyse PCNA-stained nuclei in 7 μm deparaffinised sections, with rat antibodies PCNA monoclonal antibody (dilution 1 : 100; DAKO Japan Co., Tokyo, Japan).


### 2.9. Immunohistochemical Detection of P53 Proteins

P53 proteins in the liver tissues were detected using the Avidin–Biotin Complex (ABC) method according to Tousson et al. [25] and the stained cells were extracted by colour thresholding using an Image J program to extract the brown colour for quantitation [25].


### 2.10. Statistical Analysis

To determine whether the experimental groups had any differences of significance, one-way ANOVA was employed for statistical analysis, with expression of data in the form of mean values ± SE. In the case of biochemical data, statistical significance was established, to be indicated byP<0.05. The SPSS software (version 21, SPSS® Inc., USA) was used to conduct every statistical analysis.


## 2.1. Chemicals and Drug

One kilogram of monosodium glutamate (vetsin) powder was obtained from Qingdao Huifenghe MSG Co. Ltd., China. For the MLE, freshMoringa oleifera leaves were purchased between May and June from farmers in Riyadh. The leaves were air-dried in a laboratory for five days and ground into fine particles using a simple hammer mill. The MLE was prepared by mixing one gram dried and powdered leaves with 10 ml boiling water for five minutes, according to Berkovich et al. [13].


## 2.2. Experiment Set-up and Rat Categorisation

Forty male albino rats were sourced from the animal house of the College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia, each with a weight of between 110 and 130 g and aged 9-10 weeks. The rats were kept in cages in suitable environmental conditions (22–24°C, 12-hour light/dark cycle) and were put on a diet of commercial pellet, without water restrictions. All of the experiments were conducted in compliance with the guiding principles for the care and use of laboratory animals of King Saud University in Riyadh, Saudi Arabia. The experiments were initiated 14 days after the animals were procured to allow them to become accustomed to the laboratory setting.The male rats were separated into four groups with ten rats in each, and over the four weeks of the experiments, all rats were given the same amount of food. The first group was the control group, which did not undergo any treatment. The second group was subjected to intragastrical administration of 200 mg/kg body weight of MLE over the course of four weeks. The third group was subjected to intragastrical administration of 5 mg/kg body weight vetsin powder over the course of four weeks. The fourth group was subjected to intragastrical administration of 5 mg/kg body weight vetsin powder combined with 200 mg/kg body weight MLE over the course of four weeks.When the experimental work was completed, the rats were subjected to overnight fasting. For the purposes of clinical chemistry, the rats were euthanized at the end of the fourteenth week, to permit the sampling of blood via retro-orbital puncture with the use of blood capillary tubes without heparin, in keeping with specifications. Following 10-minute incubation at room temperature, the blood samples were allowed to coagulate before being subjected to 15-minute centrifugation at 3000 rpm. After collection, the serum was isolated and stored in clean stopper plastic vials at −80°C in preparation for the examination of the serum parameters.All experimental procedures were performed in accordance with King Saud University’s ethical standards for experiments involving animals.


## 2.3. Determination of Serum Enzymes

The approach proposed by Schumann and Klauke [14] was adopted to analyse the activities of serum aspartate transaminase (AST) and alanine transaminase (ALT) with the use of a commercial kit (Human, Germany). The same kit was employed to analyse the activity of serum alkaline phosphatase (ALP) in keeping with the approach of Moss and Henderson [15]. The approach proposed by Doumas et al. [16] was followed to measure the levels of serum albumin and globulin with a commercial kit (Diamond, Egypt). Furthermore, the approach suggested by Szasz and Persijn [17] was used for approximation of GGT activity.


## 2.4. Preparation of Tissue

At the end of the experimental period, rats from each group were euthanized and subjected to a complete necropsy after 10–12 hr of fasting. Liver tissues were weighed, cut, and homogenized (10% w/v) separately in ice-cold 1.15% KCl 0.01 mol/l sodium potassium phosphate buffer (pH 7.4) in a Potter-Elvehjem type homogenizer. The homogenate was centrifuged at 10,000 g for 20 min at 4°C and the resultant supernatant was used for different enzyme assays.


## 2.5. Determination of the Biomarkers of Liver Oxidative Stress

The approach proposed by Mesbah et al. [18] was adopted to identify the composition of hepatic malondialdehyde (MDA). The approaches proposed by Aebi [19], Misra and Fridovich [20], and Habig et al. [21] were, respectively, used to identify catalase (CAT; EC 1.11.1.6) enzyme activity, superoxide dismutase (SOD; EC 1.15.1.1) enzyme activity, and glutathione S-transferase (GST; EC 2.5.1.18) enzyme activity in hepatic homogenate. Furthermore, the approach proposed by Ellman [22] helped to determine reduced glutathione (GSH) content in hepatic homogenate.


## 2.6. Analysis of Genotoxicity and DNA Damage in Hepatic Tissue

DNA damage analysis and quantification were conducted with the comet assay (single cell gel electrophoresis) method. 100 mg samples of liver were obtained from every experimental group and subjected to mincing in a chilled homogenising buffer consisting of 0.075 M NaCl and 0.024 M Na2EDTA, before undergoing gentle homogenisation with the use of a homogenizer (Ikemoto Scientific Technology Company Ltd., Japan) in ice. A suspension of cells was subjected to a 10-minute 700× g centrifugation at 4°C, followed by resuspension in cold buffer. An epifluorescence microscope permitted visualisation of the slides. The DNA migration length (comet tail length) caused by genotoxicity was established by scanning digital images in an image analyser in the case of the control and MSG groups. Observation of at least 505 arbitrarily chosen cells per sample allowed the determination of tail length (TL), representing the distance between the comet head and the final signal in the tail that could be seen, the DNA tail intensity (TI), representing the DNA fraction in the tail divided by the DNA quantity in the nucleus multiplied by 100, and the DNA tail moment (TM), representing the product of the DNA quantity in the tail and TL. Every assay was carried out three times and contrasted with control.


## 2.7. Histopathological Examination

Liver tissue samples were fixed with 10% formalin solution for 48 hours and then processed for paraffin sectioning. Sections were stained with Ehrlich’s haematoxylin and counterstaining with eosin was applied for histopathological examination according to Bancroft and Stevens [23].


## 2.8. Proliferating Cell Nuclear Antigen (PCNA) Expression

As proposed by Tousson et al. [24], an Avidin–Biotin–Peroxidase immunohistochemical method (Elite–ABC, Vector Laboratories, CA, USA) was applied to analyse PCNA-stained nuclei in 7 μm deparaffinised sections, with rat antibodies PCNA monoclonal antibody (dilution 1 : 100; DAKO Japan Co., Tokyo, Japan).


## 2.9. Immunohistochemical Detection of P53 Proteins

P53 proteins in the liver tissues were detected using the Avidin–Biotin Complex (ABC) method according to Tousson et al. [25] and the stained cells were extracted by colour thresholding using an Image J program to extract the brown colour for quantitation [25].


## 2.10. Statistical Analysis

To determine whether the experimental groups had any differences of significance, one-way ANOVA was employed for statistical analysis, with expression of data in the form of mean values ± SE. In the case of biochemical data, statistical significance was established, to be indicated byP<0.05. The SPSS software (version 21, SPSS® Inc., USA) was used to conduct every statistical analysis.


## 3. Results

### 3.1. Toxicity

Rats that were treated with the dose of MLE appeared healthy and did not show any clinical signs of disease and no mortality was recorded during the experiment duration. Neither was any mortality recorded during the experiment duration in treated rats with vetsin or with vetsin plus MLE; however, yellowish body hair and faintness were observed.


### 3.2. Markers of Liver Damage

By comparison to control, the vetsin group displayed significantly elevated levels of serum ALT, AST, ALP, and GGT (P<0.05) (Figure 1) but significantly reduced levels of serum albumin, globulin, and total protein (P<0.05) (Figure 2). In contrast, improvement in alterations in hepatic function parameters was observed in the vetsin + MLE group (Figures 1 and 2).Figure 1
The alterations in the levels of serum AST, ALT, ALP, and GGT in the four groups, each consisting of ten rats; expression of values takes the form of means SE; # and∗ denote significantly different from the vetsin group and from control, respectively.
(a)
(b)
(c)
(d)Figure 2
The alterations in the levels of serum albumin, globulin, and total proteins in the four groups, each consisting of ten rats; expression of values takes the form of means ± SE; # and∗ denote significantly different from the vetsin group and from control, respectively.
(a)
(b)
(c)


### 3.3. Oxidative Stress Markers

By comparison to the control group and the MLE group, the vetsin group exhibited significantly elevated levels of hepatic MDA (P<0.05) (Figure 3) but significantly reduced levels of hepatic GSH, SOD, catalase, and GST (P<0.05) (Figures 3 and 4). Improvement in such changes in oxidative stress markers was revealed by the vetsin + MLE group (Figures 3 and 4).Figure 3
The alterations in the levels of hepatic MDA and GSH in the four groups, each consisting of ten rats; expression of values takes the form of means ± SE; # and∗ denote significantly different from the vetsin group and from control, respectively.
(a)
(b)Figure 4
The alterations in the levels of hepatic catalase, superoxide dismutase (SOD), and glutathione S-transferase (GST) activities in the four groups, each consisting of ten rats; expression of values takes the form of means ± SE; # and∗ denote significantly different from the vetsin group and from control, respectively.
(a)
(b)
(c)


### 3.4. DNA Damage

DNA damage in hepatic tissues was analysed via a comet assay. The control and MLE groups did not differ significantly in terms of DNA damage (tail length) in hepatic tissues. In contrast, the vetsin group differed from the MLE group in that it exhibited increased tail length, tail DNA%, and tail moment, which reflected that DNA damage was significant (P<0.05). As can be seen in Table 1 and Figure 5, such extensive DNA damage was attenuated in the vetsin + MLE group by comparison to the vetsin group.Table 1
The parameters of comet assay were derived via image analysis performed on cells from every group upon experiment completion. # and∗, respectively, denote significant difference from vetsin and control.
Group
Tailed (%)
Untailed (%)
Tail length (μm)
Tail DNA%
Tail moment
Control
1.5
98.5
1.31 ± 0.17#
1.45
1.91#
MLE
2
98
1.24 ± 0.20#
1.36
1.74#
Vetsin
7
93
3.63 ± 0.26∗
3.16
10.22∗
Vetsin + MLE
5
95
2.49 ± 0.18#∗
2.04
6.55#∗Figure 5
Photomicrograph illustration of hepatic DNA damage detected in various groups via comet assay.
(a)
(b)
(c)
(d)


### 3.5. Effect of MLE Seed on Liver Histopathology

As can be seen in Figures6(a) and 6(b), hepatic sections from the control and MLE groups (G1 and G2, respectively) exhibited normal polygonal cells of large size and presenting protuberant spherical nuclei and eosinophilic cytoplasm, with a couple of spaced hepatic sinusoids interspersed among the hepatic cords with fine Kupffer cell organisation. However, hepatic sections from the vetsin group exhibited hepatotoxicity, as reflected in moderate hepatocyte deterioration, numerous apoptotic cell foci, hepatocyte cytoplasmic vacuolisation, prominent necrosis, and scattered formation of Kupffer cells among hepatocytes and encircling the portal area, as well as significant blockage of blood sinusoids (Figure 6(c)). Meanwhile, hepatic sections from the vetsin + MLE group showed hepatocytes that had undergone mild vacuolisation, a large number of apoptotic hepatocytes, relative cellular infiltration, and relative blockage in the central and portal veins (Figure 6(d)).Figure 6
Photomicrographs of liver sections in the different groups stained with haematoxylin and eosin. (a, b) Liver sections in the control and MLE groups revealed normal structure of hepatocytes (Hp) and central vein (cv). (c) Liver sections in treated rats with vetsin revealed moderate atrophied, degeneration in the hepatocytes (white arrows), marked necrosis, diffuse Kupffer cell proliferation in between some hepatocytes and surrounding the portal area (black arrows). (d) Liver sections from the vetsin + MLE group revealed mild vacuolated hepatocytes (Hp), diffuse Kupffer cell proliferation in between some hepatocytes (black arrows).
(a)
(b)
(c)
(d)


### 3.6. PCNA Expression Changes in the Liver

As Figures7(a) and 7(b) reveal, hepatic sections from the control and MLE groups exhibited slight positive response to PCNA expression in the nuclei of hepatocytes. Contrastingly, the vetsin group exhibited moderate positive response to PCNA expression in the nuclei of hepatocytes (Figure 7(c)). By comparison to control, the vetsin group showed considerably more intense PCNA expression; however, by contrast to the vetsin group, the vetsin + MLE group had significantly less intense PCNA expression in hepatic sections. The latter displayed mild positive response to PCNA expression, as is obvious in Figure 7(d).Figure 7
Photomicrographs showing PCNA staining of hepatic sections of rats from various groups. (a, b) Control and MLE groups exhibited slight positive response (arrows) to PCNA expression. (c) Liver section in the vetsin group exhibited moderate positive response (arrows) to PCNA expression, as indicated by the arrows. (d) Liver section in the vetsin + MLE group exhibited mild positive response (arrows) to PCNA expression, as indicated by the arrows.
(a)
(b)
(c)
(d)


### 3.7. P53 Expression Changes in the Liver

Negative expressions for P53 in liver sections in the control group and the MLE group were observed (Figures8(a) and 8(b)). On the other hand, strong P53expressions were observed in liver sections in rats treated with vetsin (Figure 8(c)). Hepatic sections from the vetsin + MLE group showed mild positive expressions for P53 (Figure 8(d)).Figure 8
Photomicrographs of rat liver sections in the different experimental groups stained with P53. (a, b) Faint positive reactions for P53 expression (arrows) in control and in rats treated with MLE. (c) Moderate to strong positive reactions for P53 expression (arrows) in rats treated with vetsin. (d) Mild positive reactions for P53 expression (arrows) in liver sections of rats treated with vetsin and MLE.
(a)
(b)
(c)
(d)


## 3.1. Toxicity

Rats that were treated with the dose of MLE appeared healthy and did not show any clinical signs of disease and no mortality was recorded during the experiment duration. Neither was any mortality recorded during the experiment duration in treated rats with vetsin or with vetsin plus MLE; however, yellowish body hair and faintness were observed.


## 3.2. Markers of Liver Damage

By comparison to control, the vetsin group displayed significantly elevated levels of serum ALT, AST, ALP, and GGT (P<0.05) (Figure 1) but significantly reduced levels of serum albumin, globulin, and total protein (P<0.05) (Figure 2). In contrast, improvement in alterations in hepatic function parameters was observed in the vetsin + MLE group (Figures 1 and 2).Figure 1
The alterations in the levels of serum AST, ALT, ALP, and GGT in the four groups, each consisting of ten rats; expression of values takes the form of means SE; # and∗ denote significantly different from the vetsin group and from control, respectively.
(a)
(b)
(c)
(d)Figure 2
The alterations in the levels of serum albumin, globulin, and total proteins in the four groups, each consisting of ten rats; expression of values takes the form of means ± SE; # and∗ denote significantly different from the vetsin group and from control, respectively.
(a)
(b)
(c)


## 3.3. Oxidative Stress Markers

By comparison to the control group and the MLE group, the vetsin group exhibited significantly elevated levels of hepatic MDA (P<0.05) (Figure 3) but significantly reduced levels of hepatic GSH, SOD, catalase, and GST (P<0.05) (Figures 3 and 4). Improvement in such changes in oxidative stress markers was revealed by the vetsin + MLE group (Figures 3 and 4).Figure 3
The alterations in the levels of hepatic MDA and GSH in the four groups, each consisting of ten rats; expression of values takes the form of means ± SE; # and∗ denote significantly different from the vetsin group and from control, respectively.
(a)
(b)Figure 4
The alterations in the levels of hepatic catalase, superoxide dismutase (SOD), and glutathione S-transferase (GST) activities in the four groups, each consisting of ten rats; expression of values takes the form of means ± SE; # and∗ denote significantly different from the vetsin group and from control, respectively.
(a)
(b)
(c)


## 3.4. DNA Damage

DNA damage in hepatic tissues was analysed via a comet assay. The control and MLE groups did not differ significantly in terms of DNA damage (tail length) in hepatic tissues. In contrast, the vetsin group differed from the MLE group in that it exhibited increased tail length, tail DNA%, and tail moment, which reflected that DNA damage was significant (P<0.05). As can be seen in Table 1 and Figure 5, such extensive DNA damage was attenuated in the vetsin + MLE group by comparison to the vetsin group.Table 1
The parameters of comet assay were derived via image analysis performed on cells from every group upon experiment completion. # and∗, respectively, denote significant difference from vetsin and control.
Group
Tailed (%)
Untailed (%)
Tail length (μm)
Tail DNA%
Tail moment
Control
1.5
98.5
1.31 ± 0.17#
1.45
1.91#
MLE
2
98
1.24 ± 0.20#
1.36
1.74#
Vetsin
7
93
3.63 ± 0.26∗
3.16
10.22∗
Vetsin + MLE
5
95
2.49 ± 0.18#∗
2.04
6.55#∗Figure 5
Photomicrograph illustration of hepatic DNA damage detected in various groups via comet assay.
(a)
(b)
(c)
(d)


## 3.5. Effect of MLE Seed on Liver Histopathology

As can be seen in Figures6(a) and 6(b), hepatic sections from the control and MLE groups (G1 and G2, respectively) exhibited normal polygonal cells of large size and presenting protuberant spherical nuclei and eosinophilic cytoplasm, with a couple of spaced hepatic sinusoids interspersed among the hepatic cords with fine Kupffer cell organisation. However, hepatic sections from the vetsin group exhibited hepatotoxicity, as reflected in moderate hepatocyte deterioration, numerous apoptotic cell foci, hepatocyte cytoplasmic vacuolisation, prominent necrosis, and scattered formation of Kupffer cells among hepatocytes and encircling the portal area, as well as significant blockage of blood sinusoids (Figure 6(c)). Meanwhile, hepatic sections from the vetsin + MLE group showed hepatocytes that had undergone mild vacuolisation, a large number of apoptotic hepatocytes, relative cellular infiltration, and relative blockage in the central and portal veins (Figure 6(d)).Figure 6
Photomicrographs of liver sections in the different groups stained with haematoxylin and eosin. (a, b) Liver sections in the control and MLE groups revealed normal structure of hepatocytes (Hp) and central vein (cv). (c) Liver sections in treated rats with vetsin revealed moderate atrophied, degeneration in the hepatocytes (white arrows), marked necrosis, diffuse Kupffer cell proliferation in between some hepatocytes and surrounding the portal area (black arrows). (d) Liver sections from the vetsin + MLE group revealed mild vacuolated hepatocytes (Hp), diffuse Kupffer cell proliferation in between some hepatocytes (black arrows).
(a)
(b)
(c)
(d)


## 3.6. PCNA Expression Changes in the Liver

As Figures7(a) and 7(b) reveal, hepatic sections from the control and MLE groups exhibited slight positive response to PCNA expression in the nuclei of hepatocytes. Contrastingly, the vetsin group exhibited moderate positive response to PCNA expression in the nuclei of hepatocytes (Figure 7(c)). By comparison to control, the vetsin group showed considerably more intense PCNA expression; however, by contrast to the vetsin group, the vetsin + MLE group had significantly less intense PCNA expression in hepatic sections. The latter displayed mild positive response to PCNA expression, as is obvious in Figure 7(d).Figure 7
Photomicrographs showing PCNA staining of hepatic sections of rats from various groups. (a, b) Control and MLE groups exhibited slight positive response (arrows) to PCNA expression. (c) Liver section in the vetsin group exhibited moderate positive response (arrows) to PCNA expression, as indicated by the arrows. (d) Liver section in the vetsin + MLE group exhibited mild positive response (arrows) to PCNA expression, as indicated by the arrows.
(a)
(b)
(c)
(d)


## 3.7. P53 Expression Changes in the Liver

Negative expressions for P53 in liver sections in the control group and the MLE group were observed (Figures8(a) and 8(b)). On the other hand, strong P53expressions were observed in liver sections in rats treated with vetsin (Figure 8(c)). Hepatic sections from the vetsin + MLE group showed mild positive expressions for P53 (Figure 8(d)).Figure 8
Photomicrographs of rat liver sections in the different experimental groups stained with P53. (a, b) Faint positive reactions for P53 expression (arrows) in control and in rats treated with MLE. (c) Moderate to strong positive reactions for P53 expression (arrows) in rats treated with vetsin. (d) Mild positive reactions for P53 expression (arrows) in liver sections of rats treated with vetsin and MLE.
(a)
(b)
(c)
(d)


## 4. Discussion

The aim of this study was to investigate whether vetsin-triggered hepatic toxicity, apoptosis, damage, and oxidative stress changes could be alleviated with moringa leaf extract (MLE). The results of the experiments conducted revealed that, unlike the control group, rats given vetsin exhibited, on the one hand, higher levels of ALT, AST, ALP, and GGT, which might suggest cellular leakage and functional impairment of hepatic cell membranes [26, 27], and on the other hand, decreased levels of albumin, globulin, and total proteins. Such results were consistent with those obtained by Oscar et al. [28], who concluded the occurrence of hepatocellular damage based on MSG-induced elevation in the levels of ALT and AST.There is also agreement between the present study and those by Farombi and Onyema [29] and Onyema et al. [30] with regard to the fact that rats administered with MSG had higher serum ALT levels, most likely based on the observation that hepatic oxidative stress was caused by MSG.In a similar way, Ortiz et al. [31] found that rats injected with MSG displayed increased GOT and GPT levels accompanied by hepatocyte deterioration. Indeed, the majority of cases of liver conditions are associated with increased GGT levels [32]. Hence, in this study, oxidative stress triggered by vetsin was the cause of liver damage, which in turn could have led to heightened serum GGT activity. As suggested by several authors, a number of factors might explain the disruption in hepatic enzymes, including hepatic damage, changes in cell membrane permeability, or aminotransferase heightened production or reduced catabolism [33, 34].The alterations observed in the hepatic enzymes ALT, AST, ALP, GGT, albumin, globulin, and total protein can be reversed with MLE, which has a protective effect on membrane permeability because it targets oxidants and can function as a free radical scavenger [26, 35, 36], as has been proven in previous research [35, 36]. Meanwhile, this study found that alterations in hepatic function parameters were alleviated through the administration of vetsin alongside MLE, corroborating the findings of other studies [37–40].Various pathologies are associated with involvement of free radicals, which are targeted by antioxidants, and thus help in disease prevention. Lipid peroxidation may be the reason for the cellular toxicity of vetsin. This study reported that, by comparison to control, the vetsin group had significantly higher liver MDA levels (P<0.05) and significantly lower levels of GSH, SOD, GST, and catalase (P<0.05). Thus, it could be deduced that high levels of lipid peroxidation are damaging to hepatic tissue and impair the ability of antioxidants to eliminate excess ROS production [41]. The results of this study were consistent with those of Onyema et al. [30], who observed that MSG led to a reduction in the levels of glutathione, transferase, catalase, and superoxide dismutase, lending support to the claim made by Hazar et al. [42] that liver fibrosis and deterioration occur with the involvement of oxidative stress.The abovementioned cellular transformations were attenuated by MLE, which heightened the levels of both enzymatic (SOD, CAT) and nonenzymatic antioxidants (GSH, GST), whilst decreasing the activity of MDA in hepatic tissues. The results obtained in this study are consistent with those of Uma et al. [38], who found that Moringa oleifera increased the activity of antioxidant enzymes, thus improving hepatic antioxidant status, whilst neutralising the hepatotoxic effect triggered by paracetamol. Our study is also consistent with Sharifudin et al. [36], who used rat models to investigate whether the hepatotoxic effects of acetaminophen could be neutralised by Moringa oleifera extract. The present study concluded that a period of four weeks of coadministration enabled reversal of the oxidative harm associated with vetsin-triggered liver deterioration by MLE acting as ROS scavenger.This study conducted experiments on rats to investigate the extent to which vetsin-triggered oxidative stress and genotoxicity could be counteracted byMoringa oleifera extract. The quantitative assessment of genotoxicity caused by vetsin was based on its strong ability to cause DNA damage; the study results revealed that, by comparison to the vetsin group, the vetsin + MLE group reduced DNA damage in hepatocytes. Similarly, Farombi and Onyema [29] reported that ROS production and the ensuing oxidative stress were the primary determinants of the toxic effect of monosodium glutamate.The vetsin experimental group in this study exhibited hepatocyte deterioration, significant necrosis, and scattered growth of Kupffer cells among certain hepatocytes and around the portal area, significant blockage of blood sinusoids, and elevated hepatic expression of PCNA and P53. Such observations corroborated the findings by Eweka and Om’Iniabohs [43], who found that, unlike controls, the central hepatic vein dilated under the influence of MSG. The impact of the cytotoxic substance on the liver could explain the atrophy and deterioration observed in that study. This is consistent with the current study, which observed that, unlike the control group, the central hepatic vein containing lysed red blood cells dilated as a result of vetsin.Our observations indicate that monosodium glutamate induces necrosis in rat hepatic tissues, corroborating earlier studies by Oscar et al. [28] and Ortiz et al. [31], which indicated that liver necrosis and apoptosis were caused by MSG administration. Likewise, Eweka et al. [44] identified MSG cytotoxicity as the reason for the observed hepatic atrophy and deterioration. Furthermore, this study corroborated the finding of Eweka and Om’Iniabohs [43] regarding the fact that, unlike controls, MSG induced dilation of the central hepatic vein containing lysed red blood cells.This study noted that vetsin elevated the expression of PCNA and P53 in the liver, which was suggestive of intensified proliferation and apoptosis. However, Egbuonu et al. [45] found indications that MSG triggered biliary proliferation, peribiliary fibrosis, and hepatocyte deterioration in experimental rat models. The current study identified elevated malondialdehyde (MDA) activity and reduced glutathione peroxidase synthesis in hepatic cells as the reasons for the observed liver fibrosis. Meanwhile, MSG administered orally to adult Wister rats was reported by Eweka and Om’Iniabohs [43] to cause haemolysis of red blood cells in the central vein, centrilobular haemorrhagic necrosis, loss of hepatic architectural integrity, and deterioration and hypertrophy of hepatocytes.Such effects, and its antioxidant property, mean that MLE plays an important role in the treatment of iatrogenic and toxic liver disorders [46]. Treatment with vetsin plus MLE succeeded in modulating these observed abnormalities resulting from vetsin, as indicated by the reduction of enzyme activity and the pronounced improvement of the investigated biochemical, antioxidant parameters, oxidative stress, hepatic injury, and PCNA alterations. Thus, based on such results, it can be concluded that use of vetsin is accompanied by side-effects and that the vetsin-triggered biochemical alterations, oxidative stress, liver damage, apoptosis, and PCNA modifications can be attenuated with MLE.



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