DNA Damage and Expression Profile of Genes Associated with Nephrotoxicity Induced by Butralin and Ameliorating Effect of Arabic Gum in Female Rats

Nephrotoxicity induced by exposure to environmental pollution, including herbicides, is becoming a global problem. Natural products are the prime alternative scientific research as they express better medicinal activity and minor side effects compared with a variety of synthetic drugs. This study was performed to evaluate the nephroprotective proficiency of Arabic gum against butralin-induced nephrotoxicity. Adult female rats were supplemented with Arabic gum (4.3 g/kg b.wt) and/or butralin (312 mg/L) in drinking water for 30 days. The results found that markers of serum kidney function, oxidative stress biomarkers, DNA damage, and expression of kidney specific genes (Acsm2, Ace, and Ace2) as well as histopathological examination in treated rats were conducted. Butralin-treated rats showed a rise in serum creatinine (41%), BUN (47.3%), and MDA (140.9%) as well as decrease in activity of the antioxidant markers (CAT (−21%); GPx (−70.7%); and TAC (43.2%)) in comparison with the control group. In addition, butralin treatment increased the DNA damage (221%); altered the expression levels of Acsm2, Ace, and Ace2 (−51.6%, 141.6%, and 143% respectively); and elevated histopathological lesions in the kidney tissues. Pretreatment of Arabic gum prevented butralin-prompted degenerative changes of kidney tissues. The results suggested that the protective effect provided by Arabic gum on renal tissues exposed to the herbicide butralin could be attributed to enhancement of antioxidants and increase the free radical scavenging activity in vivo.

Introduction found that treatment of rats with Arabic gum prevented gentamicin-induced lipid peroxidation in renal tissues, improved kidney function, and histological lesions [22].
As far for our knowledge goes, there are no published data with regard to the nephroprotective impact of Arabic gum against butralin intoxication. Along these lines, the current was conducted to study (a) the unfriendly impact of intense butralin exposure on rat kidney toxicity and (b) the possible mitigating effect of Arabic gum against acute butralin intoxication in rats.

Herbicide and Kits
Butralin (Amex® 48 % EC) was obtained from CFPI (Compagnie Francaise de Produits Industriels) Agro SA, France. Serum kidney function and oxidative stress kits, i.e., creatinine, urea, total protein, total antioxidant capacity, catalase, glutathione peroxidase, and lipid peroxide, were obtained from Biodiagnostic Co., Dokki, Giza, Egypt. Trizol was bought from Invitrogen (USA). The reverse transcription and PCR kits were obtained from Fermentas (USA). Arabic gum (pure) was purchased from SAGA company, Sudan. All other chemicals and reagents were obtained from tried and trusted companies.

Animals and Experimental Setup
Adult female rats weighing 136.4±5.2 g were obtained from the Animal Breeding House (ABH), National Research Center (NRC), Dokki, Giza, Egypt. Rats were preserved in specific cages, five female rats of each group (n=5), in standard food and conditions in ABH lab. All animal groups in the ABH research facility were kept as per the Guide for the Care and Use of Laboratory Animals [27], which affirmed by NRC Local Ethical Review Committee.
Female rats were partitioned into four groups as follows: group 1, animals were given water and filled in as a control group; group 2, rats got Arabic gum in drinking water at concentration 4.3 g/kg b.wt for 30 days [26]; group 3, animals got Arabic gum plus butralin (312 mg/L) in drinking water for 30 days [28]; group 4: rats got butralin (312 mg/ L) alone in drinking water related to the acceptable daily intake (ADI, 0.5 mg/kg) for rats [29]. During treatment duration, rats were investigated for any harmfulness indications and mortality day by day for 30 days.

Blood and Organ Samples
After 30 days of treatment, adult female rats were fasted overnight and blood samples were collected from retro-orbital venous plexus under anesthesia. Animals were anesthetized using ethyl ether (1.9%) and kidney tissues were removed after sacrificed by cervical dislocation. Blood samples were centrifuged (3000 rpm for 10 min at 4°C); serum was separated and stored at −20°C for biochemical studies within 4 days. After cleaning the kidney from residual blood, one part was kept in formalin (10%) for histopathological examination. A piece of the second part of kidney tissues was homogenized in phosphate-buffered saline, centrifuged for 10 min at 10,000 rpm. The supernatant was used for oxidative stress analyses. Other pieces of the kidneys were used for genetic studies.

Serum Kidney Function Biomarkers
Serum biomarkers creatinine and blood urea nitrogen (BUN) were determined by the methods of Tietz et al. [30] and Henry [31], respectively.

Comet Assay
DNA damage in treated female rats using comet assay was performed according to Blasiak et al. [36]. Slides containing kidney samples were prepared and electrophoresis (0.8 V/cm, 300mAmps) was carried out for 30 min to assess the DNA damage in 100 cells per each animal. The non-overlapping cells were randomly selected and were visually assigned a score on an arbitrary scale of 0-3 based on apparent comet tail and relative quantity of DNA in the nucleus ("class 0" exhibits normal cell and "class 3" exhibits DNA damage with a tail longer than 2× the diameter of the nucleus).

Isolation of Total RNA and Reverse Transcription Reaction
The total RNA of collecting kidney samples of treated female rats was extracted using TRIzol® extraction reagent (Invitrogen). Subsequent to the achievement of the RNA extraction, the pellets of the isolated RNA were kept in water containing DEPC. The isolated RNA was exposed to RNAse-free DNAse kit (Invitrogen, Germany) to break down the potential residues of the DNA [26]. Afterwards, the extracted RNA was divided into aliquots and kept under −80°C to use for reverse transcription.
To synthesize the cDNA copies of the kidney tissues of treated rats, synthesis kit of First Strand cDNA of Fermentas Company (RevertAidTM, MBI Fermentas) was utilized to reverse isolated RNA to cDNA through the reaction of reverse transcription (RT). The PCR program of the RT to obtain the cDNA copy was adjusted as follows: (a) 10 min at 25°C; (b) 60 min at 42°C; and (c) 5 min at 95°C. After termination of the RT reaction, the PCR tubes containing cDNA copies were kept at −20°C up to use for qRT-PCR [37].

Quantitative Real Time-PCR
The synthesized cDNA copies of the kidney tissues were used for the qRT-PCR reaction using SYBR green kit (TaKaRa, Biotech. Co. Ltd.). Melting curve was performed for each reaction and specific studied gene. The CT values of the qRT-PCR of investigating genes (Acsm2, Ace, and Ace2) were normalized to those of the GAPDH housekeeping gene ( Table 1). The quantitative values of the tested genes under investigation to the housekeeping gene were assessed using 2 −ΔΔCT method.

Histopathological Studies
The kidney was dehydrated in alcohol and fixed in paraffin wax, and thick sections were cut and stained with hematoxylin and eosin (H&E). Slides examined for histopathological alterations (10 field areas of each section) by a light microscope (Olympus BX50). The histopathological changes in kidney tissues were scored as follows: normal appearance (−), mild (+), moderate (++), and severe (+++).

Statistical Analysis
All data obtained from biochemical and molecular genetics studies were expressed as means ± SEM. The data were investigated with the Statistical Package for Social Sciences (SPSS 0.26 for Windows). The outcomes were dissected utilizing one route investigation of difference (ANOVA) trailed by Duncan's test for examination between various treatment gatherings, and statistical significance was set at P < 0.05.

Effect of Butralin and Arabic Gum in Serum Biomarkers and Kidney Weight
In the current effort, butralin was administrated to a female rat via drinking water for 30 days at a concentration of 312 mg/L corresponding to dose equal to 0.5 mg/kg based on the water intake of female rats. No mortality of toxicity has been recorded in butralin-treated female rats after 30 days of treatment. There was no significant change in food consumption or water intake during the experimental period (30 days) of all treated female rats. The relative kidney weight was significantly decreased in female rats exposed to butralin at dose 0.5 mg/kg ( Table 2). In the present study, serum creatinine and BUN were significantly increased after exposure to butralin as compared with the control group (P<0.005) ( Table 1). Pretreatment of animals with Arabic gum and concomitantly with Arabic gum significantly reduced the rise in the level of BUN and creatinine.

Oxidative Stress Biomarkers in Rats Exposed to Butralin and Treated with Arabic Gum
Butralin-treated rats showed a rise in oxidative stress (MDA) in their kidney tissues and decreased activity of the antioxidant markers (GPx, CAT, and TAC) that associated with antioxidant defense and detoxification mechanisms in hepatocytes (Table 3). Pretreatment with Arabic gum attenuated the butralin-induced oxidative damage. Hence, pretreatment with Arabic gum significantly restored the oxidative stress and elevated the total antioxidant marker activities in the group treated with Arabic gum plus butralin to the normal values.

Assessment of DNA Damage
DNA damage determination in kidney samples of female rats exposed to butralin and Arabic gum is presented in Table 4. The results explained that rate of DNA damage in kidney samples of rats treated with Arabic gum was quite similar to that in control untreated rats. However, treatment of female rats with butralin increased the rate of DNA damage with high significant differences (P < 0.01) compared with that in control rats. In contrast, treatment of female rats exposed to butralin improved the rate of DNA damage in kidney samples compared with that in female rats exposed to butralin alone.

Expression Alteration of Genes Associated with Nephrotoxicity
The expression profile of genes (Acsm2, Ace, and Ace2) involving with nephrotoxicity in female rats exposed to butralin and treated with Arabic gum is illustrated in Figs. 1, 2, and 3. Figure 1 shows that expression levels of Acsm2 gene in the kidney tissues of female rats exposed to butralin were decreased significantly compared to those in control animals. However, female rats supplemented with Arabic gum revealed high expression levels of Acsm2 gene similar to those in untreated control rats. Moreover, treatment of butralinexposed rats with Arabic gum increased significantly the levels of Acsm2 gene compared to those in female rats exposed to butralin alone. Figures 2 and 3 show that expression levels of Ace and Ace2 genes in kidney samples of female rats exposed to butralin were increased significantly compared to those in control animals. In contrast, animals supplemented with Arabic gum revealed low expression levels of Ace and Ace2 genes like those in health control rats. Furthermore, treatment of butralinexposed rats with Arabic gum decreased significantly the levels of Ace and Ace2 genes compared to those in animals exposed to butralin alone. Values are means ± SEM, n = 5; a, b, c Mean values in the same column with different superscript differ significantly ( a P < 0.01, b,c P < 0.05). The treatment with superscript "a" is statistically different when compared with treatments with superscripts "b" and "c." But the treatment with superscript "a" is not statistically different when compared with another treatment with the same superscripts "a"

Histopathological Findings
Butralin induced histological changes in kidney of female rats, including the corticomedullary junction (CMJ) and focal hemorrhages and degeneration in the tubular lining epithelium (Table 5 and Fig. 4). However, rats of control group and group treated with Arabic gum showed normal kidney tissues with the normal renal architecture formed by malpighian corpuscle which contains the glomerulus formed by capillary loops separated from the Bowman capsule by Bowman space. Also, the proximal convoluted tubules lined by simple cuboidal with microvilli as it begins at the capsule and distal convoluted tubules. The Arabic gum plus butralin treated group showed focal inflammatory cell infiltration in between the degenerated tubules in the cortex.

Discussion
In the agribusiness, utilization of herbicides to control weeds has been extensively increased worldwide. However, using these synthetic chemicals in the uncontrolled way can cause negative impacts on non-target living beings including aquatic environments [38]. In the current study, butralin was orally administrated to female rats via drinking water for 30 days at a concentration 312 mg/L which equals to dose 0.5 mg/kg based on the water intake of the Values are means ± SEM, n = 5; a, b, c Mean values in the same column with different superscript differ significantly ( a P<0.01, b,c,d P<0.05). The treatment with superscript "a" is statistically different when compared with treatments with superscripts "b" and "c." But the treatment with superscript "a" is not statistically different when compared with another treatment with the same superscripts "a" Values are means ± SEM, a, b,c Mean values in the same column with different superscript differ significantly ( a P < 0.01, b,c,d P < 0.05). The treatment with superscript "a" is statistically different when compared with treatments with superscripts "b" and "c." But the treatment with superscript "a" is not statistically different when compared with another treatment with the same superscripts "a" animals. In this study, no mortality of butralin toxicity has been recorded in exposed female rats throughout or after treatment duration. Also, there was no observed significant change in food consumption or water intake during the experimental period of all treated female rats. However, using relative kidney weight as a toxicological biomarker in this study explained that there were significant differences in female rats exposed to butralin compared with control rats. The rise in relative kidney weights could be attributed to the nephrotoxicity of butralin inducing kidney damage as reported by Mossa et al. [15]. Additionally, organ weight variations were observed by other dinitroaniline herbicides in rats and rabbits [19].
The current study showed significant elevations in serum creatinine and BUN of rats exposed to butalin as compared with the control rats. This expansion in creatinine level in female rats might be due to the impedance of the glomerular capacity and/or tubular function in the kidneys [39]. The expanded degrees of creatinine and BUN accumulation in the blood show a difficulty in the kidney function in getting rid of these substances. Refaie et al. [40] found that a raised degree of urea in blood was associated with increase of protein catabolism in the mammalian body. They suggested that BUN level is increased due to increasing the alkylated urea derivatives in rats exposed to insecticide paralethrin. Therefore, the current study suggests that expansion of urea and creatinine levels in the serum of female rats is associated with histological alterations inducing a reduction in the glomerular filtration rate as a result of butralin intoxication.
Oxidative stress is a vital sign for understanding the mechanism of pesticide toxicity because it is associated with alteration of oxidant/antioxidant status and defense pathway [16,41]. In this study, butralin-exposed rats showed a rise in MDA (as an oxidative stress marker) and decrease in activity levels of the antioxidant markers (GPx, CAT, and TAC). A

Control
Arabic gum Butralin Arabic gum+Butralin Rrelative expression of Acsm2/ GAPDH) Treatment Fig. 1 Expression levels of Acsm2 gene in kidney samples from female rats exposed to butralin and treated with Arabic gum. Data are presented as mean ± SEM. The treatments with a,b,c followed by different superscripts are significantly different (P < 0.05). The treatment with superscript "a" is statistically different when compared with treatments with superscripts "b" and "c." But the treatment with superscript "a" is not statistically different when compared with another treatment with the same superscripts "a" previous study reported that paraquat (as it related to dinitroaniline) reduces the activity levels of GPx, and CAT [42]. Female rats exposed to butralin increased significantly the rate of DNA damage compared with that in control rats. Our previous work indicated that the ROS formation was raised significantly with the exposure to butralin in a dose-dependent manner [28]. It can be suggested that butralin-motivated oxidative stress induces mitochondrial function deterioration, protein synthesis and assembly, DNA damage, and slow, incremental kidney cell damage and eventual kidney dysfunction through the generation of ROS in exposed rats. The elevated levels of reactive oxygen species and their related oxidative stress in kidney cells can lead to kidney cell apoptosis and necrosis. Naturally, free radicals are created during cellular biological reactions in equilibrium with the biological antioxidant systems [43,44].
Acsm2 gene is considered a kidney-specific "KS" gene in mammals [45,46]. They reported that Acsm2 gene is expressed when the function of renal tissues became mature in kidneys of rats. Moreover, Gomez et al. [47] and Watanabe et al. [46] indicated that Acsm2 expression is likely associated with the growth and maturation alterations occurring in the kidney cells after few weeks' postpartum rats and mice. In the present work, we found that the expression levels of Acsm2 gene were reduced in rats exposed to butralin. This reduction in Acsm2 levels might be associated with the histopathological lesions in proximal tubules induced by butralin as observed in our study. Ace function regulates RAS (renin-angiotensin system) which is coinciding with renal disease [48][49][50]. The RAS system is working in the breakdown of the AngII profibrotic peptide into Ang-1-7 [51,52]. So, Ace and Ace2 genes are greatly expressed  Fig. 2 Expression levels of Ace gene in kidney samples from female rats exposed to butralin and treated with Arabic gum. Data are presented as mean ± SEM. The treatments with a,b,c followed by different superscripts are significantly different (P < 0.05). The treatment with superscript "a" is statistically different when compared with treatments with superscripts "b" and "c." But the treatment with superscript "c" is not statistically different when compared with another treatment with the same superscripts "c" in the diseased kidney [51]. In the same line, the current results found that rats exposed to butralin showed low expression levels of Ace and Ace2 genes compared with control rats.
Pretreatment with Arabic gum attenuated the toxic effect of butralin-induced oxidative damage in female rats. Arabic gum used in the African' traditional therapy as beneficial natural prebiotic dietary supplement decreases blood pressure and attenuates the progression of chronic kidney diseases. The useful effects of Arabic gum can be explained on its antioxidant and antiinflammatory activity in kidney tissues [11,53,54]. Hence, pretreatment with Arabic gum significantly restored the GPx, CAT, and TAC levels near to those in control rat's values. Several studies indicated that Arabic gum has been found to act as an antioxidant product in addition to its useful effects on kidney function [11,54,55]. The present results found that Arabic gum exhibited  Fig. 3 Expression levels of Ace2 gene in kidney samples from female rats exposed to butralin and treated with Arabic gum. Data are presented as mean ± SEM. The treatments with a,b,c followed by different superscripts are significantly different (P < 0.05). The treatment with superscript "a" is statistically different when compared with treatments with superscripts "b" and "c." But the treatment with superscript "c" is not statistically different when compared with another treatment with the same superscripts "c" Table 5 Histopathological changes in the kidney tissue of female rats exposed to butralin and the protective effect of Arabic gum, based on scoring severity of injury significant reduction in MDA and increase in GPx, CAT, and TAC activities in kidney tissue of the Arabic gum plus butralin group. These results are consistent with the previous evidence of Al Za'abi et al. [56], who proposed the main mechanism for the beneficial action of Arabic gum in adenine-induced kidney disease through its antioxidant properties. The antioxidation induced by Arabic gum might be one of the most likely mechanisms supplying to its beneficial effect against kidney injury. This antioxidant effect of Arabic gum was established previously by in vitro studies, which showed that Arabic gum had a dose-dependent scavenging of free radicals produced enzymatically and nonenzymatically [57]. Hence, Arabic gum could have the ability to inhibit ROS generation induced by herbicides [10,11] due to suppression of MDA. Therefore, it could be suggested that Arabic gum scavenges butralin free-radical generation and, in turn, inhibits MDA-induced DNA damage in renal tissues, which has been recommended to protect renal tissue damage induced by butralin. Further, Arabic gum could ameliorate mitochondrial function deterioration and DNA double strand breaks. The present results indicated that the expression profile of Acsm2, Ace, and Ace2 genes is altered in rats exposed to butralin, which is likely attributed to the damage occurred in renal tissues. So, renal tissue protection by treatment of Arabic gum could be also associated with modulated expression alteration of Acsm2, Ace, and Ace2 genes induced by butralin. Therefore, the protective effect provided by Arabic gum on renal tissues exposed to the herbicide butralin could be attributed to the enhancement of antioxidants and increase the free radical scavenging activity in vivo.

Conclusion
In vivo exposure to butralin induced MDA, decreased antioxidant enzyme activities, elevated DNA damage, and changed the mRNA levels of kidney-specific genes, which is likely imputed to the damage occurred in renal tissues. These negative actions of butralin on kidney tissues could be attributed to increase free radical formation as reported in our previous work [28]. Treatment of female rats with Arabic gum suppressed the toxicity effects induced by butralin. So, the protective effect provided by Arabic gum on renal tissues exposed to the herbicide butralin could be attributed to the enhancement of antioxidants and increase the free radical scavenging activity in vivo.