Scholarly article on topic 'Amelioration of liver and kidney functions disorders induced by sodium nitrate in rats using wheat germ oil'

Amelioration of liver and kidney functions disorders induced by sodium nitrate in rats using wheat germ oil Academic research paper on "Chemical sciences"

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Abstract of research paper on Chemical sciences, author of scientific article — M.M. Anwar, N.E. Mohamed

Abstract The purpose of this study was to evaluate the effect of sodium nitrate administration on some biochemical parameters and to explore the ability of Wheat germ oil (WGO) as a natural source of antioxidants to minimize the deleterious effects of sodium nitrate. The results showed significant increase in alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and significant decrease in butyryl cholinestersea (BChE) content in hepatic tissue in nitrate group as compared to control and wheat germ oil groups through the experimental period. Furthermore, there was a significant increase in thiobarbituric reactive substances (TBARS) accompanied by significant decrease in reduced glutathione (GSH) content in rat renal tissue after 28 and 42 days of treatment with drinking water containing sodium nitrate. Significant decrease was also observed in serum estradiol (E2) in group treated with nitrate through the experimental period. In addition, microscopically examination of renal tissue showed atrophy of glomerular tuft and congestion of renal blood vessels in nitrate treated group. Administration of WGO to rats with sodium nitrate suggesting role of WGO as a natural protective antioxidant agent in hepatic and renal tissues. WGO also stimulates estrogen secretion and inhibits oxidative damage that may be attributed to the presence of biologically active components (unsaturated fatty acids, unsaponifible matters and sterols matters) as antioxidant and cyto-protective activities. It can be concluded that WGO offers a great advantage for therapeutic purpose to minimized sodium nitrate free radical induced cell damage.

Academic research paper on topic "Amelioration of liver and kidney functions disorders induced by sodium nitrate in rats using wheat germ oil"

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Amelioration of liver and kidney functions disorders induced by sodium nitrate in rats using wheat germ oil

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M.M. Anwar a, N.E. Mohamed b

a Plant Research Department, Nuclear Research Center, Atomic Energy Authority, P. O. 13759, Cairo, Egypt b Biological Applications Department, Nuclear Research Center, Atomic Energy Authority, P. O. 13759, Cairo, Egypt

ARTICLE INFO

ABSTRACT

Article history: Received 9 October 2014 Received in revised form 26 November 2014 Accepted 26 November 2014 Available online 13 December 2014

Keywords: Nitrate

Wheat germ oil Oxidative stress Estrogen Rats

The purpose of this study was to evaluate the effect of sodium nitrate administration on some biochemical parameters and to explore the ability of Wheat germ oil (WGO) as a natural source of antioxidants to minimize the deleterious effects of sodium nitrate.

The results showed significant increase in alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and significant decrease in butyryl chol-inestersea (BChE) content in hepatic tissue in nitrate group as compared to control and wheat germ oil groups through the experimental period. Furthermore, there was a significant increase in thiobarbituric reactive substances (TBARS) accompanied by significant decrease in reduced glutathione (GSH) content in rat renal tissue after 28 and 42 days of treatment with drinking water containing sodium nitrate. Significant decrease was also observed in serum estradiol (E2) in group treated with nitrate through the experimental period. In addition, microscopically examination of renal tissue showed atrophy of glomerular tuft and congestion of renal blood vessels in nitrate treated group. Administration of WGO to rats with sodium nitrate suggesting role of WGO as a natural protective antioxidant agent in hepatic and renal tissues. WGO also stimulates estrogen secretion and inhibits oxidative damage that may be attributed to the presence of biologically active components (unsaturated fatty acids, unsaponifible matters and sterols matters) as anti-oxidant and cyto-protective activities.

It can be concluded that WGO offers a great advantage for therapeutic purpose to minimized sodium nitrate free radical induced cell damage.

Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/3.0/).

* Corresponding author. E-mail addresses: nelshahat@yahoo.com, naglaa22000@yahoo.com (N.E. Mohamed). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications. http://dx.doi.org/10.1016/jjrras.2014.11.004

1687-8507/Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction

In recent years, considerable attention has been paid to the problem of nitrate due to the intensive use of nitrates as agricultural fertilizers which reach to humans and animals by different routs (Manassaram, Backer, & Moll, 2006; Mande et al., 2012). Nitrate is a naturally occurring form of nitrogen and is an integral part of the nitrogen cycle in the environment. It is formed from fertilizers, decaying plants, manure and other organic residues. Nitrate is found in air, soil, water, and vegetables food and is produced naturally within the human body (Ogur et al., 2005). In fact certain foodstuffs such as maize, guinea corn, carrots, potatoes, sunflower, pumpkins and cabbage are known to accumulate large quantity of nitrates even at the normal fertilizer application rate of 150 kg/ ha (Awodi, Ayo, Nwude, & Dzenda, 2005; Manassaram et al., 2006). It is also used as a food additive, mainly as a preservative and antimicrobial agent (Speijers & van den Brandt, 2003). Due to the increased use of synthetic nitrogen fertilizers and livestock manure in intensive agriculture, vegetables and drinking water may contain higher concentrations of nitrate than in the past (Manassaram et al.,2006).

The major source of nitrate in the human body is through intake of food and water (IPCS, 1999). Vegetables may account for more than 70% of the nitrates in a typical human diet (ATSDR, 2001). Drinking water may contain variable amounts of nitrates which accounts for up to 21% of total nitrates intake in a typical human diet (Manassaram et al., 2006). The presence of nitrate in vegetable as in water and generally in other foods is a serious threat to man's health. Nitrate per se is relatively non toxic (Mensinga, Speijers, & Meulenbelt, 2003), but approximately 5% of all ingested nitrate is converted by microflora in the gastrointestinal tract to the more toxic nitrite (Pannala et al., 2003). Nitrite and N- nitroso compounds which form when nitrite binds to other substances before or after ingestion are toxic and can lead to severe pathologies in humans (Speijers & van den Brandt, 2003). The ability of animals to resist the toxic effects of environmental agents is dependent on the detoxication and antioxidant systems. Recently, several nutrients and other chemicals are effective antioxidants, such as vitamins, trace elements, amino acids and their derivatives, fatty acids and plant phenolics (Ayo, Minka, & Mamman, 2006; Son, Mo, Rhee, & Pyo, 2004; Suteu et al., 2007).

WGO is extracted from the germ of the wheat kernel and is particularly high in policosanol contents specially octacosanol (Irmak & Dunford, 2005) which has been shown to increase physical performance (Kim, Park, Han, & Park, 2003) to be helpful in cholesterol management, chronic inflammatory reactions and neurological disorders (Reddy et al., 2000). Therefore, in recent decades WGO has received much attention in treatment of diseases involving oxidative damage (Blommers, Elisabeth, Deklerk, Piter, &Maiyer, 2002). WGO is a valuable source of essential fatty acids including linoleic acid and alpha lionlenic acid which maybe beneficial by increasing endurance, lowering cholesterol levels, and assisting muscular dystrophies and other neuromuscular disorders. WGO may be also changed the intensity of lipid peroxidation processes by stimulating the tocopherol redox-system (Leenhardt, Fardet, Lyan, Gueux, & Remesy, 2008).

WGO is a rich source of natural antioxidant toco-pherols and sterols and also is a rich source of B complex vitamins which may have significant implications in chemoprevention (Jensen, Koh-Banerjee, Hu, Franz, & Sampson, 2004; Lui, 2007). In addition, WGO is a source of easily assailable vitamin E which acts as an inhibitor of oxidation processes in body tissues and protects cells against the effects of free radicals which are potentially damagingby products of the body's metabolism. It is well know that free radicals can cause cell damage that may contribute to the development of cancer (Traber, Vitamin, Shil, Olson, & Shike, 1999). Moreover, WGO not only prevents autoxidation of unsaturated fatty acids but also, generates DNA protective properties (Gelmeza, Kineal, & Yener, 2009).

In view of this consideration, the current study has been designed to investigate the effect of nitrate in a short term experiment (for 42 days i.e 6 weeks) on albino rats and the role of WGO as an antioxidant to counteract the toxic effect of nitrates was taken into consideration.

2. Material and methods

2.1. Experimental animal

Forty eight female albino rats weighing 130 ± 10 g obtained from the animal house of Biological Applications Department, Nuclear Research Center, Atomic Energy Authority, Cairo, Egypt were used in experiment. Animals were maintained under normal requirement ventilation, illumination conditions and adequate stable balanced diet and water.

2.2. Wheat germ oil preparation

Wheat germ oil (WGO) was packaged as a liquid and was purchased from Arab company for Pharm-Medicinial plants, (MEPACO) Egypt. WGO was given to animals by gavages using stomach tube and was prepared as water emulsion (1.0 ml emulsion 900 mg wheat germ oil/Kg body weight/rat).

2.3. Experimental design

Animals were randomly divided into 4 equal groups (n = 12)

1 Group 1: Control group: rats received 1.0 ml tap water through stomach tube (placebo treatment) through the experimental period.

2 Group 2: Wheat germ oil group: rats received WGO orally (900 mg WGO/Kg body weight/rat) for five consequence days per week through the experimental periods (28 and 42 days) (Mohamed & Anwar, 2010).

3 Group 3: Sodium nitrate group: rats were supplied with 500 mg sodium nitrate/L in drinking tap water every day through the experimental period (Mohamed & Anwar, 2010; Zaki et al., 2004). Sodium nitrate (NaNo3) was produced from Sigma Chemicals Company, Egypt.

4 Group 4: Wheat germ oil plus sodium nitrate group: rats were supplied with 500 mg sodium nitrate/L in drinking tap water and treated with 900 mg WGO/Kg body weight/rat for five consequence days per week through the experimental periods.

Six animals from each group were randomly sacrificed 24 h after termination of treatment processing. Blood samples were withdrawn by cardiac puncture after light anesthesia. Sera were then separated and kept frozen for subsequent biochemical analysis. The liver and one kidney were taken, washed with saline solution, weighed then homogenized in phosphate buffer (pH 7.4) to yield 10% homogenate. The other kidney was dissected out and imbedded immediately in 10% formalin for histo pathological examination.

2.4. Biochemical analysis

Determination of alanine transaminase (ALT) and aspartate transaminase (AST) activities, alkaline phosphatase (ALP) and butyrylcholinesterase (BChE) were determined in hepatic tissue by the method of Reitman and Frankel (1957), Kind and King (1954) and Knedel and Bottger (1967) respectively. Estimation of thiobarbituric acid reactive substance (TBARS) and reduced glutathione (GSH) content in the kidney homogenates were determined according to the methods of (Beutler, Duran, & Kelly, 1963; Yoshioka, Kawada, Shimada, & Mori, 1979) respectively. Serum estra-diol (E2) was estimated using commercial radioimmuno-assay (RIA) kits, purchased from Immunotech A Beckman Coulter Company. The level of estradiol represents the mean levels of eostrous cycle.

2.5. Histological preparation

For the histological preparation, animals were dissected to remove kidney through the experimental period. Kidney tissues were cut into small pieces and then fixed in 10% neutral buffered formalin. The tissue was routinely processed and sectioned at 5-6 mm thickness with a microtome and stained with haematoxylin and eosin for histo-pathological studies.

2.6. Statistical analysis

The data were expressed as mean ± standard error for 6 animals. Results of control and the experimental groups were statistically analyzed using one way analysis of variance (ANOVA) according to Snedecor and Cochran (1982).

Differences between means of treatments were tested according to Duncan (1955).

3. Results

3.1. Effect of administration ofWGO with sodium nitrate on liver function

The data in Table 1 showed that daily administration of WGO with sodium nitrate significantly decreased the elevation of ALT, AST and ALP and increased BChE as compared to the corresponding values of sodium nitrate treatment.

3.2. Effect of administration ofWGO with sodium nitrate on TBARS, GSH and serum estradiol

It was noticed from Table 2 that administration of sodium nitrate in drinking water resulted in significant increase in TBARS and significant decrease in GSH content in renal tissue as compared to the control and wheat germ oil groups. The data showed also that the daily administration of wheat germ oil with sodium nitrate significantly decrease the elevation of renal TBARS and increased GSH content. Table 2 revealed also that nitrate treatment in drinking water caused a significant decrease in serum estradiol (E2) level as compared to the control group at both time intervals.

3.3. Effect of administration ofWGO with sodium nitrate on kidney histo-pathological examination

Microscopically, kidney of rat from control group revealed normal histological structure (Plate 1: Fig. 1). Similarly, kidney of rat from WGO group for 28 and 42 days of administration revealed no histo-pathological changes (Plate 1: Fig. 2 and 5).

On the other hand, kidney of rat from group administrated sodium nitrate for 28 days showed congestion of renal blood vessels and some sections showed congestion of inter-tubular renal blood vessels (Plate 1: Fig. 3). After 42 days of treatment, renal tissue of rat showed focal renal hemorrhage and some sections showed vacillations of renal tubular epithelium and atrophy of some glomerular tuft (Plate 1:Fig. 6). Some sections from group 4 of rats administrated with WGO with sodium nitrate for 28 days showed no histo-pathological changes (Plate 1: Fig. 4). After 42 days of treatment congestion of renal blood vessel was the only histo-pathological finding observed

Table 1 - Alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and butyryl cholinestersea (BChE) levels in hepatic homogenate rats of four experimental groups.

Parameters Experimental Control WGO Nitrate Nitrate + WGO

periods (days)

ALT U/g wet tissue 28 10.27 ± 1.51c 11.40 ± 1.06c 19.90 ± 0.72a 15.65 ± 1.0b

42 12.00 ± 0.99c 12.73 ± 0.99bc 18.80 ± 1.14a 15.13 ± 0.58b

AST U/g wet tissue 28 9.16 ± 1.18b 10.26 ± 0.92b 16.92 ± 1.13a 11.13 ± 0.64b

42 12.46 ± 0.66b 11.82 ± 0.86b 21.33 ± 1.78a 12.27 ± 0.58b

ALP IU/g wet tissue 28 8.89 ± 3.13b 10.55 ± 2.88b 29.61 ± 2.81a 14.44 ± 1.33b

42 7.82 ± 1.48b 7.97 ± 1.61b 24.86 ± 5.52a 10.20 ± 2.22b

Butyryl cholinesterase 28 36.06 ± 1.73a 37.55 ± 0.87a 23.77 ± 1.14c 30.12 ± 0.64b

U/g wet tissue 42 39.18 ± 0.99a 40.27 ± 1.32a 22.89 ± 1.62c 35.19 ± 1.11b

Data presented mean values ± S.E. in particular groups of 6 rats.

Values in the same row with different superscripts differ significantly (P < 0.05).

Table 2 - Thiobarbituric acid reactive substance (TBARS), reduced glutathione (GSH) contents in kidney homogenate and serum estradiol in adult female rats of four experimental groups.

Parameters Experimental periods (days) Control WGO Nitrate Nitrate + WGO

TBARS nmol/g wet tissue 28 13.08 ± 1.05c 13.97 ± 1.24c 24.21 ± 1.04a 17.26 ± 0.59b

42 13.97 ± 1.08c 10.68 ± 0.75d 26.99 ± 0.76a 15.84 ± 0.88b

GSH mg/g wet tissue 28 20.37 ± 1.46b 24.11 ± 0.85a 11.80 ± 0.80d 18.01 ± 0.86b

42 22.26 ± 0.90b 28.86 ± 1.97a 10.68 ± 0.74d 15.70 ± 0.91c

Serum E2 (ng/ml) 28 19.20 ± 0.37a 18.02 ± 0.66ab 17.56 ± 0.62b 16.81 ± 0.23cb

42 16.89 ± 0.79a 16.93 ± 0.36a 13.69 ± 0.39b 17.91 ± 0.75a

Data presented mean values ± S.E. in particular groups of 6 rats.

Values in the same row with different superscripts differ significantly (P < 0.05).

in renal tissue of rat treated with WGO and sodium nitrate (Plate 1: Fig. 7).

4. Discussion

4.1. Effect of administration of WGO with sodium nitrate on liver function

Nitrate salt is partially converted to nitrite by oral bacteria and by stomach acids, helping to reduce gastrointestinal tract

infection (Combs, 2000). Nitrate is in high concentration in drinking water and meat products which induced methemo-globinemia in infinite. It has been implicated in the formation of methemoglobin and carcinogenic nitrosamine in human (Chan, 2011; Kapor, 2004). Information available shows that nitrates and nitrites are both oxidation products and ready sources of NO, that NO reacts rapidly with superoxide to form highly reactive peroxynitrite (ONOO~) and such products may increase lipid peroxidation (LPO) which can be harmful to different organs including kidney (Hassan et al., 2009; Rocha et al., 2012).

Plate 1 - Light photomicrographs of kidney sections of rats (H and E X 400). Fig. (1): Control. Fig. (2): WGO group (28 days). Fig. (3): Sodium nitrate group (28 days). Fig. (4): WGO + sodium nitrate group (28 days). Fig. (5): WGO group (42 days). Fig. (6): sodium nitrate group (42 days). Fig. (7): WGO + sodium nitrate group (42 days).

Antioxidant supplementation may become more important when intake from the diet is insufficient or where the production of oxidative free radicals is greater. Butyryl cholinesterase (BChE) is one class of cholinesterase enzyme which is synthesized by the liver and is abundant in the serum (Massoulie, Pezzementi, Bon, Krejci, & Vallette, 1993). The decrease in BChE is used as an indicator of liver disorders (Ogunkeye & Roluga, 2006). This hypothesis is supported by the increase in AST, ALT and ALP activities in hepatic tissue in treated group with nitrate.

According to the result of this study, WGO has protective properties that can prevent undesired consequences of repeated nitrate administration on ALT, AST, ALP and BChE in hepatic tissue. This finding is in agreement with to the finding of (Mohamed & Anwar, 2010) who recorded that the intake of WGO ameliorating the effect of nitrate. Co-administration of WGO with nitrate compensated the imbalance of the AST, ALT, ALP and BChE in hepatic tissue caused by sodium nitrate to the control level. This may be attributed to that WGO is a valuable source high amount of unsaturated fatty acid and high amount of unsapounfible matter special sterols and essential fatty acids including lenoleic acid and alpha linolic acid which stimulate fatty acid oxidation (Vecera et al., 2003) as well as octacosanol and natural vitamin E (Gu et al., 2002).

4.2. Effect of administration of WGO with sodium nitrate on TBARS, GSH and serum estradiol

The present results revealed significant increase in TBARS and significant decrease in GSH in renal tissue in treated group with nitrate. This results became in accordance with Farombi and Onyema (2006) and Mohamed and Anwar (2010). One of the establish mechanisms of toxicity of nitrate is their ability to induce oxidative stress through the generation of free radicals (Manassaram, Backer, & Moll, 2007), therefore, the significant increase in TBARS may be attributed to the damaging effects of free radicals which were initiated by the peroxidation of poly unsaturated fatty acids of blood lipo-protein, phospholipids and cholesterol ester moiety of cell membrane. This oxidation damage of different organs was reflected by the accumulation of abnormal products (Microalwski, 1994). Nitric oxide (NO) an unstable molecule is synthesized from L-arginine by the enzyme nitric oxide syn-thase and reacts with oxygen species and biological molecules to form a varity of end products including nitrite, nitrate radicals and S-nitrosothiols (Mehta et al., 2009).

In the present study treatment with WGO alone revealed non significant changes in the investigated parameters indicating its safe in use. WGO is a source of easily assimilable vitamin E which represented by 89.3% and acts as inhibitor of oxidation processes in body tissues (Abd El-Aziem, Abdou, & Nasr, 2005). Vitamin E has antioxidant and free radical scavenging activities, which suggests that this vitamin may modulate oxidative DNA damage in mammalian cells (Jacobs, Connell, Rodriguez, & Seymour, 2001). Vitamin E neutralizes the increased ROS production induced by toxic substances (Nielsen, Skionsberg, & Lyberg, 2008). Vitamin E is the primary liposoluble antioxidant which may have an important role in scavenging free oxygen radicals and in stabilizing the cell

membranes thus maintaining its permeability (Navarro, Arroyo, Martin, Bello, & Villalba, 1999).

Co-administration with WGO cause significant decrease in TBARS and significant increase in GSH content in renal tissue. The obtained results became in accordance with Alessandri et al. (2006), Hargrove, Greenspan, and Hartle (2004) and Singh, Li, and Porter (2006) who reported that WGO decreases oxidative stress probably due to vitamin E in WGO which is a potent peroxyl radical scavenger that prevents the propagation of free radical damage in biological cell membranes (Leppala et al., 2000). The intake of WGO resulted in a rapid increase in vitamin E content in the brain, liver, heart, lungs, kidneys and spleen (Paranich, Cherevko, Frolova, & Paranich, 2000). The obtained results are in agreement with that of Alessandri et al. (2006) who reported that WGO decrease the oxidative stress, so it protects the renal cellular structure.

Nitrate treatment in drinking water caused a significant decrease in serum estradiol (E2) level as compared to control. However, the estradiol level presented herein recorded the means which is the average of estradiol concentration at the different stages of oestrous cycles. The administration of WGO in combination with nitrate exhibited a significant increase in the level of E2 at both two time intervals. This improvement attributed to the content of WGO of vitamin E (Mohamed & Anwar, 2010; Paranich et al., 2000) reported that WGO consists of refractive index 1.4780, acid value 2.13 mg KOH/g oil, peroxide value 8.0 meq/oil, iodine number 133.5 and saponification number 184, fatty acids composition of WGO consisted of unsaturated fatty acid (80.0%) (palmitic oil, stearic, oleic, linoleic, linolenic) and total saturated fatty acid (19.72%), unsaponifiable matter components of WGO composed of 77.0% hydrocarbons and 23.0% total sterols. It contains the other plant materials such as phytosterols (Lewis et al., 2003; Nakari, 2005) which have estrogenic activity and can alter cholesterol metabolism (Ostlund, Racette, Okeke, & Stenson, 2002). However, cholesterol is a major precursor of the ovarian hormones synthesis. Co-administration of WGO with nitrate eliminated the effect of nitrate on estradiol hormone as well as enzymes and this results are in accordance with Mehranjani, Abnosi, Naderi, and Mahmodi (2007).

4.3. Effect of administration ofWGO with sodium nitrate on kidney histo-pathological examination

In the present study, it was found that sodium nitrate causes atrophy of glomerular tuft and congestion of renal blood vessels which may be due to nitric oxide formations, which causes vascular smooth muscle relaxation. This leads to dilatations of their lumens and increases their blood flow.

Finally, the present study reveals toxic effects of sodium nitrate on the liver and renal functions. Therefore, more researches must be done on other organs of the body to highlight its effects on these organs.

5. Conclusion

It could be concluded that supplementation of WGO can protect against free radical damage induced by sodium nitrate and improve liver and kidney functions.

Acknowledgment

The authors acknowledge the expert assistance rendered by Prof. Dr K. A. Ahmed, Professor of Veterinary Medicine, Cairo University for performing histo-pathoilogical examinations and interpretations.

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