Evaluation of ameliorating role of avocado Persea americana fruit extract against monosodium glutamate-induced toxicity in pregnant female albino rats and their offspring

Background : Although monosodium glutamate (MSG) is commonly used as a food additive, the application of higher doses or prolonged uses significantly leads to accumulations in living cells and finally produces cellular toxicity. Persea Americana (avocado) has recently gained substantial popularity and is often marketed as a “superfood” because of its unique nutritional composition, antioxidant content, and biochemical profile . Aim : To evaluate the potential ameliorative role of avocado fruit extract against MSG-induced nephrotoxicity in pregnant rats and their offspring. Thirty-two (24 females and 8 males) albino rats were used in this study. After an acclimatization period of two weeks; the animals were mated, and the pregnant rats were randomly divided into four groups; control (G1), avocado (G2): they were supplemented with 50 mg/kg b.w. of avocado fruit extract, MSG (G3): they were given 3g / kg b.w. of MSG, every other day, and MSG &Avocado (G4): they were given an oral dose of MSG alternatively with avocado fruit extract. At the end of weaning, the female rats and their offspring were sacrificed and the blood was collected and the kidneys were excised to evaluate the renal biochemical and histopathological, and immunohistochemical investigations. Results: In MSG-treated mothers’ rats, the renal cortical sections displayed severe histopathological lesions including little renal corpuscles, atrophied glomeruli, and relatively wide Bowman ' s space. However, the offspring displayed mild renal histopathological lesions compared with their mothers. The immunohistochemical results revealed strong PCNA and Bax expression in the renal tissues of MSG-exposed mother rats and their offspring if compared with the control. Furthermore, the mean percentage value of positively expressed cells for caspase-3 appeared significantly higher in the renal cells of MSG-induced mother's rats and their offspring if compared with the control. Additionally, the levels of serum antioxidants (SOD&CAT) and potassium ions appeared significantly lowered while the level of MDA, urea, and creatinine appeared significantly higher if compared with the control. Co-supplementation of avocado fruit extract to MSG-induced mothers rats and their pups successfully alleviated the histopathological, immune-histo-chemical, apoptotic as well as biochemical changes caused by MSG. Conclusion: Avocado fruit extract has a powerful ameliorative role against MSG-induced renal toxicity in mother rats and their offspring.


Introduction:
Preclinical studies have connected MSG consumption to hepatotoxicity. There have been claims that MSG usage has an impact on lymphocyte apoptosis [1].
MSG-fed rats have been proven to exhibit gonadal dysfunction. The kidneys are harmed by the overuse of MSG, as has been shown [2,3]. In addition to humans, other animal species have also been reported to transmit flavors [4]. It has been demonstrated that a variety of types decrease when fruits are consumed.
Numerous diseases are less common when fruits are consumed [5,6]. For moms throughout the perinatal period, several of the important elements found in fruits are advised [7]. Fruits contain bioactive chemicals and are whole-food sources of magnesium [8]. All forms of food typically contain additives [9].
People were thought to be at risk from food additives [10]. The Acceptable Daily Intake (ADI) control method provides support for the evaluation of food additives [11]. The Food and Drug Administration (FDA) was established to safeguard consumers against any negative health impacts [12]. It is described as the additive's numerical value [13]. The highest concentration of an additive that is functionally beneficial in a food or food category is referred to as the maximum use dose [10]. To determine the possible harmful effects of a food additive these additives must be subjected to trials for an appropriate toxicity assessment [14].

Material and methods:
Monosodium glutamate (MSG) in the form of white crystals (99% purity) was purchased from the local market of Damanhur city, Egypt. Preparation of Avocado (Persea Americana) fruit Extracts. Fresh avocado pear fruits were purchased from the local market in Damanhour city, Egypt. The plant was identified as Persea americana (Lauraceae). Avocado fruit was extracted according to the method of Biglari et al. [15]. For this study, thirty-two albino rats (24 females and 8 males) weighing 180-200 g (8-9 weeks) were obtained from the Holding Company for Biological Products and Vaccines (VACSERA, Cairo, Egypt). Rats were mated in the special matting cages

Experimental groups:
The pregnant rats were randomly divided into four groups as follows, six for each group (n=6). Group I (control): they received an oral dose of 1% carboxymethyl cellulose, every other day. Group II (Avocado): they were given a daily oral dose of Avocado fruit extract at a dose of 50 mg/kg b.w.
Biglari et al. [15] dissolved in 1% carboxy-methyl cellulose (CMC), every other day from the 4 th day of gestation till the end of weaning. Group III (MSG): they were given an oral dose of MSG at 3g / kg b.w.
Contini et al. [16], every other day, from the 4 th day of gestation till the end of weaning. Group IV (MSG &Avocado): they were given an oral dose of MSG alternatively with avocado fruit extract by the same previous doses.
Sample collection and tissue preparation: At the end of the experimental period (end of weaning = 21days postnatal), the mother's rats and their offspring were sacrificed to collect the blood and then dissected immediately to remove the kidneys. Histological techniques for Haematoxylin and Eosin stains were done according to Bancroft and Gamble [17].
Immunohistochemical staining technique: The demonstration of antigens by immunohistochemistry (IHC) is a two-step process involving first, the binding of a primary antibody to the antigen of interest, and second, the detection of bound antibody by a chromogen. The primary antibody may be used in IHC Adekomi [18]. Creatinine Analyzer-2 (Beckman Coulter Inc., USA) in combination with a specific kit of reagents (Hichem Creatine Pak, Elan Diagnostics, USA) was applied to estimate serum creatinine content in blood samples [18]. Catalase (CAT) and superoxide dismutase (SOD) were assayed according to previous studies [19].

Histopathological observations
In the control as well as in the Avocado extract

Immunohistochemical observations
Immunohistochemical expression of PCNA: The renal cortical sections from control and avocado extract-supplemented groups of mother rats and their offspring displayed weak to moderate PCNA expression. However, a strong positive expression of PCNA appeared in the cortical sections of MSGinduced mothers' rats. On treatment with avocado fruit extract in MSG-induced mother's rats, the immunoreactivity of PCNA appeared moderately expressed but still higher than control. Generally, the expression of PCNA appeared prominent in the cells of cortical tubules rather than the glomerulus (figures3&4).

Changes in caspase-3 activity in the renal tissues
The obtained flow cytometric data revealed that the mean percentage value of caspase-3 activity was significantly higher in the renal cells of MSG-induced mother's rats and their offspring (73%, 60.1%) if compared with their control (39.7%, 36.2%) respectively. On treatment with avocado fruit extract after MSG induction, the mean percentage value of caspase-3 activity in the renal cells was significantly declined (mothers' rats =52.6%, Offspring =52.1%) if compared with MSG-induced group but still significantly higher than control (figure7).

Changes in serum antioxidants (CAT, SOD, and GPx) and MDA
In MSG-induced mothers' rats and their offspring,  (Table 2 and figures 12-17).

Changes in serum electrolytes:
The  (Table 3 &figures                           Metabolites of the drugs that are excreted by the kidney may also cause cellular damage leading to kidney dysfunction [22]. Several studies in animals have shown that MSG is toxic to various organs such as the liver [23]. Other studies revealed that if female rats are exposed to MSG during the gestation or lactation period this leads to deleterious developmental changes in their offspring [24]. Persea americana has gained substantial popularity [25]. The current work is mainly designed to evaluate the potential therapeutic role of avocado fruit extract against MSG-induced nephrotoxicity in pregnant rats and their neonates. The results of the present work revealed that exposure of pregnant rats to MSG during the lactation period is followed by pronounced histopathological lesions in the renal cortical sections of both mother rats and their offspring.
In mother rats, these renal lesions included atrophied glomeruli. In the offspring, the histopathological lesions were represented by atrophied glomeruli.
The obtained results go parallel with the findings of previous reports on adult rats [26]. Another report revealed that MSG can induce changes in the renal cytoarchitecture, and increase glomerular hypercellularity [27]. The histopathological signs that appeared in the renal tissues of offspring maternally induced with MSG go parallel with the findings of Bhattacharya & Ghosh [28] who found dilated urinary space in mice neonates maternally induced with MSG.
Previous research confirmed that exposure to an overdose of MSG is implicated in the liberation of free reactive oxygen species [29]. Furthermore, MSG supplementation by oral intake has been shown to alter lipid peroxidation byproducts [30]. Paul et al. [30] found reduced activities of SOD and CAT in the Previous reports declared that avocado is a good source of unsaturated fatty acids [32]. Avocado is rich in other natural antioxidants as phenolic compounds [33].
The present study showed that the administration of MSG to pregnant rats is implicated in the induction of renal dysfunction as evidenced by the increased levels of serum creatinine in both mother rats and their offspring. These findings are in agreement with the studies of [34] who reported that prolonged administration of MSG can induce alteration in the glomerular. Adejuwon and Adokiye [35] added that serum creatinine level relates to glomerular function. The significant increase in creatinine content of the serum following the administration of MSG may be attributed to the compromise of the renal functional capacity [36].
Tawfik and Al-Badr [37] added that exposure to MSG can harm renal function which might be due to oxidative stress induced by MSG on the renal tissue.
In MSG-administered groups and co-supplemented with avocado fruit extract revealed an improvement in renal functions. These findings go parallel with the study of Mahadeva et al. [38] who reported that avocado fruit acts as a nephroprotective agent. An avocado oil-rich diet has been shown to modify renal functions [39]. The data of the present study revealed that in MSG-exposed mother rats and their offspring the levels of serum sodium ions appeared significantly higher while the level of potassium ions appeared significantly lowered if compared with the control.
The data concerned with significant elevation of serum sodium in MSG-exposed rats go parallel with the finding of Ilegbedion et al. [40]   apoptosis [45]. Caspase-3 is a pro-apoptotic marker that accelerates programmed cell death. This study showed that the administration of MSG led to significant increases in Bax and caspase-3. This was in the same line with Sarhan et al. [46] who explained that glutamate induced the Ca 2+ influx and destruction of the internal mitochondrial membrane potential [47].
Avocado fruit extract was found to decrease the higher levels of caspase 3, and Bax caused by MSG. Roset et al. [48] revealed an increase in caspase-3 activity in the rats treated with MSG in the testicular tissues.
Schelman et al. [49] indicated that glutamate receptors may be the result of apoptosis depending on the  [52]. The diabetic rats displayed glomerular shrinkage, tubular casts, and inflammatory cellular infiltration [52]. Rats on a high-fat diet for two weeks displayed more inflammatory cells, cardiac muscle fibers in decline, and round and clogged blood arteries [53].