PROTECTIVE ROLE OF AQUEOUS EXTRACT OF ATRIPLEX HALIMUS L. AGAINST BENZENE-INDUCED DAMAGE ON RENAL FUNCTION AND GLOMERULAR CELLS IN RATS

Objective: Benzene (BZ) is an important pollutant compound, present in both occupational and general environment that has been linked to adverse health effect. The aim of the current study was to investigate the curative and preventive effect of Atriplex halimus L. (Ah) extract against BZ-induced nephrotoxicity in rats. Methods: A total of 30 male albino Wistar rats were divided into five groups (n=6): Control, Ah, BZ, AhP+BZ, and BZ+AhC. BZ (100 mg/kg b.w) was added in their drinking water for 15 weeks. Ah (Aqueous Extract of aerial parts) was given by gavage during the past 30 days of BZ-exposed for curative treatment (AhC) and all the duration of BZ exposure in the animals for preventive treatment (AhP). Some biochemical, oxidative stress parameters, and histopathology of kidney tissue were studied. Results: Obtained results revealed that BZ exposure to rats caused a significant elevation in urea, creatinine, and malondialdehyde levels. Then, it led to reducing the glutathione level, catalase, and glutathione-S-transferase activities when compared to the control animals. Histopathological studies showed a massive damage in kidneys of the BZ-exposed rat. Our results showed that curative treatment of aqueous extract of Ah has a partial correction of the biochemical, oxidative stress parameters, and kidney morphology, but preventive treatment has a dramatic effect on the previous parameters. Conclusion: Results demonstrated the beneficial effects of Ah preventive treatment against stress oxidative and kidney damage induced by BZ.


INTRODUCTION
Benzene (BZ) with C6H6 formula is the primary aromatic compound [1]. It has been extensively used since 100 years and is still widely used as a solvent and intermediate compound [2] and produced in large quantities in the chemical and fuel industry including gasoline, automobile exhaust, several polymers, resins, synthetic fibers, and cigarette smoke [3]. It is also used in manufacturing products such as rubber, detergents, lubricants, pesticides, and drugs [4]. Workers employed in industries that manufacture or use BZ is exposed to its highest levels [5]. BZ is a ubiquitous environmental pollutant [6] which affects human health includes hematological diseases, reproductive system, respiratory problems, kidney disease, liver injury, and central nervous system [7]. Thus, humans are unavoidably exposed to BZ both occupational and environmental settings and which poses a serious risk to public health [8].
Toxicity of BZ most likely results from oxidative metabolism of BZ to reactive products [9]. These reactive products can lead to the formation of reactive oxygen species (ROS), which can directly damage pivotal molecular targets, such as DNA, proteins, and lipids, leading to the formation of new compounds and modified structures [10], and in this condition; oxidative stress has a critical role in the pathophysiology of several kidney diseases [11,12].
The association between food and health is well established. Recent overwhelming attention to plant products and traditional medicine has focused on natural antioxidant for preventing the tissues from various xenobiotics induced toxicities [13,14]. Atriplex halimus L. (Ah) is a famous halophytic shrub for the nutritive and energetic value, not only for livestock but also as food for nomads and the local steppe population.
The young shoots of Guettaf are eaten by the man, preparing its like spinach [15]. Ah also has always been a popular folk remedy for treating hyperglycemia, stomach painkiller, and intestinal worms [16]. In light, these considerations, the aim of the present study is to determine the effects of Ah against BZ-induced nephrotoxicity in rats.

Zeghib and Boutlelis
sample of the aqueous extract of Ah (0.2 ml) and 1 ml of 1:10 diluted Folin-Ciocalteu reagent were added after 4 min to 800 µl of saturated sodium carbonate (75 g/l). After 2 h of incubation at room temperature, the absorbance was measured at 765 nm. The tests were carried out 3 times to ensure the reproducibility of the results [18]. The total phenolic content was expressed in mg equivalent of gallic acid per gram of sample.

Estimation of condensed tannin
The tannins content in the extract was determined spectrophotometrically according to Broadhurst and Jones [19]. Catechin was used to make the calibration curve. 0.5 ml of sample pipetted into a tube in aluminum foil, and mixed with 3.0 ml of vanillin reagent (Freshly prepared 4% w/v of vanillin in methanol), and then added 1.5 ml of concentrated hydrochloric acid and mixed thoroughly. After 15 min of reaction at 20-2°C, the absorbance was measured against water at 500 nm.

Animals and treatment
A total of 30 adult male albino rats, weighing 227.14±3.42 g, were taken from the animal house of Pasteur Institute, Algeria. They were placed in five groups of six rats in each and kept in animal's house of Molecular and Cellular Biology Department, University of El-Oued, Algeria. Standard rat food and tap water were available ad libitum for the duration of the experiments. Animals were adapted for 2 weeks under the same laboratory conditions of photoperiod (12 h light/12 h dark) with relative humidity 62% and room temperature of 24±1°C. The experimental procedures were carried out according to the National Institute of Health Guidelines for Animal Care and approved by the Ethics Committee of our Institution. The adult Wistar albino rats were randomly divided into five groups, each containing 6 rats as follow: • Group 1 was orally received normal water and serving as a control. • Group 2 was received oral gavage 200 mg/kg b.w (3 days/week) aqueous extracts of Ah for 15 weeks. • Group 3 treated by 100 mg/kg, bw/day of BZ (Fig. 1)  During treatment, body weight was recorded periodically during the experiment weeks.

Blood collection and preparation of tissue samples
At the end of treatment, rats were fasted for 16 h, anesthetized with chloroform by inhalation, rats were decapitated, and blood samples were transferred into ice-cold centrifuge tubes. The serum was prepared by centrifugation for 10 min at 3000 revolutions/min and utilized for urea, creatinine, albumin, uric acid, and total proteins concentrations, and lactate dehydrogenase (LDH) activity assays. Blood glucose was measured by glucometer. The kidney was rapidly excised, washed, weighed, and stored at −20°C for oxidative stress parameters analysis. Another part of the kidney was excised for histopathological examination.

Measurement of biochemical parameters
Serum urea, creatinine, albumin, uric acid, and total proteins concentrations, and LDH activity were measured using commercial kits obtained from Spinreact (Barcelona, Spain).

Preparation of homogenates
About 1 g of the kidney was homogenized in 9 ml of buffer solution of Tris buffer saline (TBS, pH=7.4). Homogenates were centrifuged at 9000× g for 30 min at 4°C, and the obtained supernatant was used for the determination of oxidative stress markers.

Determination of malondialdehyde (MDA) level
MDA was measured according to the method described by Quintanilha et al. [21]. The MDA reagent (trichloroacetic acid, thiobarbituric acid, and hydrochloric acid) was added to aliquots of the homogenate mixed with 2% (w/v) ethanolic solution of butylated hydroxytoluene. Then, the mixture was heated for 15 min in a boiling water bath. After cooling, the precipitate was removed by centrifugation, and the absorbance was measured at 532 nm. The results were expressed as nmol of MDA/mg protein.

Determination of reduced glutathione (GSH) level
GSH concentration was performed with the method described by Ellman (1959) based on the development of yellow color when DTNB is added to compounds containing sulfhydryl groups. In brief, 0.8 ml of tissue homogenate was added to 0.2 ml of 0.25% sulfosalicylic acid and tubes were centrifuged at 2500 g for 15 min. Supernatant (0.5 ml) was mixed with 0.025 ml of 0.01 M DTNB and 1 ml TBS (pH 7.4). Finally, the absorbance at 412 nm was recorded. Total GSH content was expressed as nmol GSH/g tissue [22].

Determination of glutathione-S-transferase (GST) activity
GST activity of tissues was measured spectrophotometrically by the method of Habig et al. [23]. Using CDNB as an electrophilic substrate that binds to GSH with the participation of the enzyme and forms a colored GSH-substrate complex, detected at 340 nm. The activity of GST was expressed in terms of μmol CDNB-GSH conjugate formed/min/g tissue.

Determination of catalase (CAT) activity
CAT activity was assayed by the decomposition of hydrogen peroxide according to Aebi method, and the absorbance of the sample was measured at 240 nm for 1 min using a UV-spectrophotometer. The enzymatic activity was calculated in terms of the international unit/min/g of tissue [24].

Protein level assay
The protein content of the supernatant was spectrophotometrically estimated by Bradford method using bovine serum albumin as standard [25].

Histopathological study
After rats sacrificed, the part of kidney tissues was removed and immersed in fixative (solution 10% formaldehyde) until the time of slices preparation. It was dehydrated in ascending graded series of ethanol, cleaned with toluene, and immersed in paraffin. Sections of 4~6 Μm were prepared from paraffin blocks using a Leica Rotary Microtome (Wetzlar, Germany), and colored with hematoxylin and eosin. Histopathological evaluation was performed with a light microscope.

Statistical analysis
Data were carried out using Student's t-test to compare means among the groups. Differences were considered statically significant at p<0.05.

Phenolic and tannin compounds
Total phenolic and condensed tannin compounds were expressed in terms of gallic acid equivalents (mg of GAE/g sample) and of catechin equivalents (mg of Ca/g sample), respectively, using the Fig. 1: Chemical structure of benzene [20] Zeghib and Boutlelis following equation based on the calibration curve: Y=0.0104x+0.0819, R2= 0.9925 for phenolic compounds and Y=0.003x+0.006 R2=0.9882 for condensed tannin compounds. Total phenolic and condensed tannin contents of aqueous Ah extract were represented in Table 1.
Body and relative kidney weight BZ exposure rats caused a decrease in body weight gain (p<0.001) and an increase in relative kidney weight (p<0.01) compared to the control rats. Preventive Ah treatment of BZ exposure rats (AhP+BZ) was totally restored the body weight gain (p<0.05) and relative kidney weight (p<0.05) level as compared to the BZ rats. Curative treatment of the extract aqueous of Ah was significantly (p<0.05) increase body weight gain compared to BZ rats, but no effect for on relative kidney weight in (BZ+AhC) rats compared to BZ rats. No effect on body weight gain and relative kidney weight of Ah alone treatment in rats compared to the control rats (Table 2). Tables 3 and 4, BZ exposure caused a significant increase (p<0.05) in serum creatinine and urea level and a significant decrease in serum protein (p<0.01) levels. Furthermore, no effect of Ah alone or BZ on blood glucose, blood ratio urea/creatinine, serum albumin concentration and LDH activity compared to control. Meanwhile, Ah made a recovery in the above-mentioned biochemical parameters for preventive treatment (AhP+BZ) but the low effect for curative treatment (BZ+AhC). In another way, treatment with the aqueous extract of Ah alone has no effect on most of these parameters.

Oxidative stress parameters
Results presented in Table 5 show a significant increase (p<0.05) in MDA level and a significant decrease in GSH concentration (p<0.01), GST (p<0.01), and CAT (p<0.001) activities in BZ group compared to the corresponding control values. Treatment with Ah partially restored the MDA levels (p<0.001), CAT (p<0.05), and GST (p<0.05) activities

Zeghib and Boutlelis
for curative effect (BZ+AhC) and totally restored the MDA (p<0.01), GSH (p<0.05) levels and CAT (p<0.01), and GST (p<0.05) activities for preventive effect (AhP+BZ). In addition, treatment with the plant extract alone does not cause any change in oxidative stress parameters.

Correlations between analyzed parameters
The results in Table 6 represent the correlation between serum kidney markers (creatinine and urea) and kidney oxidative stress markers (MDA, GSH, GST, and CAT) for experimental groups. Our results showed that there was a significant positive correlation (p<0.05) between serum creatinine concentration and kidney MDA level. However, there was a significant negative correlation between serum creatinine concentration and kidney antioxidative markers GSH (p<0.05), GST (p<0.001), and CAT (p<0.01). In addition, there was a significant positive correlation (p<0.05) between serum urea concentration and kidney MDA level and a negative correlation between serum urea concentration with GSH (p<0.01) level and GST (p<0.01) activity, but there was no significant correlation between serum urea and kidney CAT activity.

Histopathological studies
Microscopic observation of histological sections of kidney from the control and Ah treated rat showed normal renal parenchyma with glomeruli, tubules and normal cortical, and medullary areas ( Fig. 2a  and b). However, histological sections of the BZ exposed rat kidney revealed severe degeneration represented by necrosis and glomerular hypertrophy apparent to their architecture with tubular necrosis, dilatation, and tubular vacuolation. In addition, hemorrhage, foci of inflammation and large space of Bowman, was observed in the kidney sections of this group (Fig. 2c). Kidney section of (AhP+BZ) groups when rats received the aqueous extract of Atriplex protective treatment with BZ indicated protection from tubule and glomerulus (Fig. 2e). Atriplex curative treatment partially decreased severe damage to kidney tissue (Fig. 2d). The histopathological changes are graded and summarized in Table 7. Histological grading was made according to four severity grades: − (none), + (mild), ++ (moderate), and +++ (severe).

DISCUSSION
The finding of this study indicates that BZ-induced nephrotoxicity in the rat through the increase relative kidney weight, serum urea and creatinine levels, and alters renal antioxidant and oxidative stress parameters and also caused histopathological alterations in kidney tissue. However, our study indicated that Ah has a dramatically preventive effect on BZ-induced nephrotoxicity through the potential restoration of biochemical and histopathological parameters in kidney tissue rat. As well, in a curative mode, Ah partially restored the renal damage, suggesting that Atriplex exhibits inhibitory action thus causing nephroprotection.
BZ exposure at a dose (100 mg/kg) resulted in a significant decrease in body weight and increase in relative kidney weight. Ibrahim et al. reported a significant decline in the body weight gain of the animals intoxicated with BZ. Moreover, according to him; this reduced of the body weights may be due to the overall increased degradation of lipids and proteins as a result of the direct effects of BZ [26]. Then, cotreatment of Atriplex with BZ restored the weight of the body and kidney as normal, which can indicate the prevention against the injury effect of BZ on kidney as evidenced macroscopically.
The increased levels of urea and creatinine are considered as the marker of impaired kidney functions [27,28]. Thus, in our study, an increase of plasma urea and creatinine reflected the diagnosis of renal damage. These results were inconsistent with the study of Khan and Yusufi [29]. On other, occupational exposure to BZ in petrol stations was showed that serum creatinine was significantly higher in the exposed group than in the unexposed employees [30]. Aqueous extract of Ah preventive treatment decreased the raised serum urea and creatinine and maintained healthy kidney function, but low effect, we observed for curative treatment in these parameters. Moreover, BZ orally administrated to rats caused a significant decrease in serum protein concentration and less increase in serum LDH activity. However, BZ   Recent studies reported that BZ-initiated toxicity has been linked to the production of ROS [31]. More specifically BZ can be enzymatically bioactivated to reactive metabolites (catechol, benzoquinones, hydroquinone, and phenol), which can produce ROS, decreases antioxidant activity, and hence increases oxidative stress [32,33]. Kidney is one of the major tissue sites of BZ's metabolites and BZ's conjugated metabolites for phenol, catechol, and hydroquinone, phenyl sulfate and hydroquinone glucuronide, and muconic acid [34,35]. These compounds form covalent adducts with diverse macromolecules such as nucleic acids and proteins in the kidney [36].
MDA is one of the major lipid peroxidation products and is used as a marker of cell membrane destruction and tissue damage [37]. Our data demonstrated that BZ administration caused a significant increase in MDA levels as compared to control group in the kidney tissue, which reflects extensive lipid peroxidation process in the kidney of rats after BZ exposure. Our results are in good agreement with Çavuşoğlu et al. who found that BZ administration at a dose 50 mg/kg of body weight for 50 days caused a significant increase in kidney MDA [38].
Results from the present study demonstrate that the levels of endogenous antioxidants both enzymatic and non-enzymatic (GSH and CAT, GST) are significantly lowered in the BZ-treated group compared to the normal control rats, which are supported by the findings of Verma and Rana [39] who reported that BZ administration decreased the level of GSH in rats kidney. Furthermore, Hetal [40] demonstrated that BZ exposure leads to a decrease in antioxidant enzymes activity. The sulfhydryl reduced GSH is a small intracellular thiol (-SH) molecule, play an essential role in the detoxification processes [41]. Furthermore, previous studies indicated that GSH depletion is one of the important consequences of toxic injury; which might be consumed during the metabolic reaction of BZ [42]. CAT and GST antioxidant enzymes play a critical role in the protection of tissues from the deleterious effects of BZ. GST represent a major group of detoxifying enzymes [43], which catalyze the detoxification of BZ oxepin, metabolite potential reactive, and carcinogenic of BZ through conjugation with GSH and excretion in the urine as S-phenylmercapturic acid [44]. Finally, as an H2O2 scavenger, CAT has been shown to play an important role in the protection of cells against toxicity from the hydroquinone metabolite [45]. Therefore, the decreased activities of these antioxidant enzymes indicated a failure of the antioxidant defense system in BZ group, may have been due to oxidation reactions initiated by BZ metabolites and the influx of ROS induced by BZ exposure.
Extract Ah treatment, (especially preventive treatment) dramatically decrease MDA level and increase GSH, CAT, and GST level in the BZ group. This effect can be attributed to the activity of its antioxidants and the ROS scavenging capacity. In this study, aqueous extract of Ah contains higher levels of total phenolic and condensed tannin. Many researchers reported a significantly high correlation between the high antioxidant activity of plant extracts and its high total phenolic compounds. Further, according to the previous research, Ah contain a wide range of bioactive metabolites. It is a source of Vitamins A, C, and D, polyphenols, flavonoids, tannins, alkaloids, saponins, and resins [46]. These constituents are good indicators for the antioxidant activity of this plant that may be involved in the prevention of cellular damage and many human diseases, caused by free radicals [47,48]. These antioxidants molecules interact with free radicals and terminate the chain reaction before vital molecules are damaged. They donate an electron to stabilize a free radical [49]. Therefore, it may be suggested that Ah treatment can reduce the BZ toxicity by decreasing the oxidative stress through the activation of antioxidant defense systems, the ROS scavenging and the inhibition of LPO in BZ administered rats. These observations might indicate that Atriplex has protective effects on BZinduced oxidative stress and it may potentially reduce the unfavorable effects of BZ in the kidney.
Histopathological observation of renal tissue sections from the BZ group confirmed the biochemical results and showed clear signs of nephrotoxicity. Stained sections reveal they marked severe glomerular and tubular degeneration, glomeruli hypertrophy, loss of brush border membrane, vascular congestion, inflammatory cell infiltration, vacuolization of tubular cells, tubular dilatation, and increased number of pyknotic nuclei. Overproduction of ROS in chronic BZ exposure can cause damage of important biomolecules (proteins, lipids, and DNA) and increase intra-renal pro-inflammatory and fibrogenic factors, which can lead to cell death (necrotic) and chronic inflammation associated with tissue renal damage [50]. However, Atriplex improved the renal histological changes with normal glomeruli and tubules in preventive effect. This protective effect of the extract may be attributed to the presence of antioxidant and anti-inflammatory components in this plant.

CONCLUSION
The current study demonstrates that the administration of Ah can reduce (or completely prevent) several toxicities induced by BZ in kidneys. This protective potential may involve the powerful antioxidant properties of this plant by eliminating free radicals and oxidative damages caused by BZ. Hence, dietary supplementation with Ah could be a useful method to protect populations at high risk of environmental and/or occupational BZ chronic intoxication, and for scavenging the free radicals generated in the kidney.

ACKNOWLEDGMENTS
The first author would like to thank the Faculty of Sciences of Nature and Life, University of El Oued, Algeria, for the permission to utilize the institute facilities to make this work.

AUTHOR'S CONTRIBUTIONS
The conception of the study, drafting the article and interpretation of data was done by Khaoula Zeghib author; Djahra Ali Boutlelis author was responsible for the acquisition of data and analysis.