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EFFECT OF VITAMIN C ON BISPHENOL A INDUCED HEPATO& NEPHROTOXICITY IN ALBINO RATS

Marcelle R. Haroun,M.D.*, Ibrahim S. Zamzam, M.D.*,Eslam S. Metwally, M.D.*, and Rabab Sh. EL-Shafey, M.S.C. * *Forensic Medicine & Clinical Toxicology Department, Faculty of Medicine, Benha University, Egypt [email protected] [email protected] [email protected] [email protected]

ABSTRACT Bisphenol-A (BPA) is a worldwide used endocrine disruptor that is incorporated in many plastic industries. The exposure of humans to that substance starts early during the fetal life. Many agencies raised warnings against the excessive use of such substances. The present study aimed to evaluate the oxidative stress effect of BPA on liver and kidney of albino rats, and whether co-administration of vitamin C can ameliorate this oxidative damage. Albino rats were divided into six groups: -ve control group,+ve control group,vitamin C (60mglkg) treated group, BPA treated group, BPA+ high dose of vitamin C (60mglkg) treated group, and BPA+ low dose of vitamin C (5.5mglkg) treated group The oxidative stress arising from BPA was evaluated in liver and kidney tissues. In addition, serum creatinine, uric acid levels, (AST) and (ALT) activities as markers of kidney and liver function were measured. Biochemical analysis revealed significant reduction in activities of enzymatic antioxidants in BPA treated group and BPA + vitamin C treated groups in high & low doses as compared to control group. In addition, there were significant increase in AST, ALT, uric acid, and creatinine levels in BPA treated group and reduction in their concentrations BPA + vitamin C treated groups in high & low doses as compared to control group. Histopathological and ultrastructure changes of liver and kidney were observed in BPA treated group and BPA + vitamin C treated groups as compared to control group. The present study demonstrated that BPA could induce hepato& nephrotoxicity, affecting oxidant/antioxidant balance in rat liver and kidney. In addition, Co-administration of vitamin C in low dose (5.5 mg/kg) didn't prevent this oxidative damage. While co-administration of vitamin C in high dose (60 mg/kg) augmented this oxidative damage. Keywords: Bisphenol A, Vitamin C, Oxidative stress, andantioxidants.

INTRODUCTION used as lacquers to coat metal products BPA is an endocrine disrupting such as food cans, bottle tops, and chemical which is used mainly as a water supply pipes. In addition, BPA is monomer in the production of used in the production of other types of polycarbonate plastics (PC) and as a resins, flame-retardants, and in the precursor of epoxy resins. processing of polyvinyl chloride plastic Polycarbonate plastics are used in food and in the manufacturing of thermal and drink containers, the resins are paper. Some polymers used in dental

Egypt J. Forensic Sci. Appli. Toxicol Vol 16 (2) December suppl 2016 Haroun et al. 58 sealant and tooth coatings contain BPA prevent cell and tissue damage that as listed in reports of the European could lead to diseases (Halliwell, Commission’s former European 1999). But, with the excessive Chemicals Bureau (ECB, 2008).The production of ROS for any reason, the ubiquitous and extensive use of BPA oxidative damage occurs in the cells. containing products results in a high Also, many environmental human exposure worldwide contaminants have been reported to (Vandenberg et al., 2010) . disrupt the prooxidant/antioxidant the primary endocrine disrupting balance of cells by inducing oxidative activities of BPA extend beyond its stress (Ho et al., 1998). ability to mimic, enhance or inhibit the The majority of studies on BPA activity of endogenous estrogens and/or have focused on its endocrine disrupt estrogen nuclear hormone disrupting and potential adverse effects receptor action, and include effects on the developing reproductive system. upon the androgen systems (Sun et al., However, there is only limited 2006), disruption of thyroid hormone information concerning the effects of it function (Hamers et al., 2006), diverse on other tissues, so that the present influences on development, study aimed to evaluate the oxidative differentiation and function of the stress effect of BPA on liver and central nervous system (Miyatake et kidney and whether co-administration al., 2006), and influences on the of vitamin C can ameliorate this immune system (Tian et al., 2003). oxidative damage in albino rats. BPA can cause liver, kidneys, brain, and other organs injury by MATERIAL & METHODS formingreactive oxygen species (ROS). Chemicals used Reactive oxygen species are cytotoxic BisphenolA (BPA) powder (97%) agents causing oxidative damage by purity was purchased from attacking cell membrane and DNA. SigmaAldrich Company (USA). Antioxidants are scavengers by Vitamin C powder (100%) purity preventing cell and tissue damage that was purchased from ELGomhureia could lead to diseases (Kabuto et al., pharmaceutical and chemicalcompany, 2004). Cairo, Egypt. Vitamin C is an important dietary Animals and Experimental antioxidant which significantly Design decreases the adverse effects of ROS Experimental Animals: Seventy formed in the cell. Many biochemicals, two adult albino rats weighing 180-200 clinical and epidemiologic studies have g were maintained on stock diet and indicated that vitamin C may be of kept under fixed appropriate conditions benefit in chronic diseases such as of housing and handling. All cardiovascular disease, cancer, and experiments were carried out in cataract, probably through antioxidant accordance with the research protocols mechanisms (Carr and Frei, 1999). according to the Ethics Committee of Antioxidants neutralize ROS by Scientific Research, Faculty of donating one of their own electrons, Medicine, Banha University which ending the electron "stealing" reaction; followed the recommendations of the they act as scavengers, helping to National Institutes of Health Guide for

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Care and Use of Laboratory Animals extrapolating the therapeutic dose to rat (Publication No. 85-23, revised 1985). dose on the basis of body surface area The albino rat was the animal of ratio (conversion factor 0.018 for rats) choice for the experiments. This is by referring to the table of “Paget & because the anatomy as well as Barnes" (Paget and Barnes, 1964). i.e. physiology of these animals is closely For rats, Humans dose x 0.018 = X related to that of human and most of g/200gm of rat. So rat dose = 1.08 chemical and biochemical experimental mg/200gm of rat/ day. Conversion to studies were carried out with these dose/kg body wt. = 1.08 mg × 5 = animals often produce similar results 5.4mg / kg (nearly about 5.5 mg/kg). with those conducted on human The selection of these doses was (Ogunleye and Omotoso, 2005). based on the previous study done by Experimental Design: The Korkmaz et al., (2010). animals were equally divided into six All animals were received the groups:- drugs orally. The oral administration (1) Group I (negative was via appropriate sized metallic control group): lifted without cannula dressed with plastic cover that intervention to measure the basic exceeds the tip by 2-3 mm to prevent parameters. injury of the esophagus. (2) Group II (positive At the end of the experiment the control group): Each rat was treated animals were sacrificed and subjected with olive oil (vehicle of bisphenol A to the following. & vitamin C) once daily, orally for six A) Biochemical study: weeks. Preparation of serum: (3) Group III (vitamin C Blood samples were taken from treated group): each rat was treated their hearts by 5ml syringes. Blood with vitamin C (60mg/kg/day) samples were left to coagulate at room dissolved in olive oil, once daily, temperature, then centrifuged at 986 g orally, for six weeks. for 15 minutes, the clear non- (4) Group IV (bisphenol A hemolyzed supernatant serum was treated group): Each rat was treated quickly removed and used for the with bisphenol A (25 mg/kg/day) estimation of serum enzymes (AST, dissolved in olive oil, once daily, ALT) , uric acid and creatinine. orally, for six weeks. Handling of tissue samples: (5) Group V (treated with The liver and kidney of each bisphenol A and high dose of vitamin animal was quickly removed, weighed C): Each rat was treated orally with and handled as following:- bisphenol A (25 mg/kg/ day) and 1- A portion of both organs was vitamin C (60 mg /kg/ day) once daily, dissected and rapidly frozen until orally, for six weeks. analyzed. The liver is homogenized in (6) Group VI (treated with 10 ml and kidney in 6ml of ice cold bisphenol A and low dose of vitamin phosphate buffer (50 mM pH 7.4, 0.1% C): Each rat was treated orally with triton X and 0.5 mM EDTA). The bisphenol A (25 mg/kg/ day) and homogenates were centrifuged at 1753 vitamin C (5.5 mg /kg/ day). This dose g for 15 min at 4°C using a high speed of vitamin C was calculated by cooling centrifuge (Type 3K-30,

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Sigma, Osterode-am-Harz, Germany). 30 min. Thereafter, 10% Triton X-100 The clear supernatants were separated was added to have a final concentration and used for analysis of oxidative stress of 1%. The sample, thus obtained, was parameters. used to determine the catalase activity 2- Another portion of both organs by measuring the breakdown of was dissected and settled in 10% H2O2spectrophotometrically at 240 nm formalin for histopathological (Mukherjee et al., 2010) examination. 3- Glutathione peroxidase (GPX) : 3- The rest of both organs were The method proposed by Reddy et settled in 4% glutaraldehye for electron al. (1995) was adopted for assaying the microscopic examination. activity of peroxidase. In the presence . Oxidative stress parameters of hydrogen donor pyrogallol or assessment: dianisidine, peroxidase converts H2O2 Lipid peroxidation to H2O and O2. The oxidation of Lipid peroxidation was assayed by pyrogallol or dianisidine to a coloured determining the level of product called purpurogalli can be malondialdehyde (MDA) by measuring followed spectrophotometrically at thiobarbituric reactive species using the 430nm. method of Chattopadhyay et . Assay of liver enzymes al.,(2003). The thiobarbituric acid 1- Alanine aminotransferase reactive substances react with (ALT):- thiobarbituric acid to produce a pink Glutamate pyruvate transaminase colored complex, which can be read (GPT) level was calculated colorimetriclly at 532 nm. spectrophotometrically using the . Assay of antioxidant enzymes commercial test of GPT (ALT) with 1- Superoxide dismutase (SOD);- spin lab (Spinreact company), Spain. SOD was assayed according to the 2- Aspartate aminotransferase method of Kakkar et al. (1984).The (AST):- assay of SOD is based on the inhibition Glutamate oxaloacetate of formation of NADH- transaminase (GOT) level was phenazinemethosulfate – calculated spectrophotometrically using nitrobluetetrazoliumformazon. The the commercial test of GOT (AST) colour formed at the end of the reaction with spin lab (Spinreact company), can be extracted into butanol and Spain. measured at 560nm. . Assay of renal functions 2- Catalase (CAT): 1- Creatinine:- Catalase activity was assayed by Creatinie level was calculated measuring the breakdown of hydrogen spectrophotometrically using the peroxide (H2O2). The weighed amounts commercial test of CREATINIE-J with of the hepatic tissue were homogenized spin lab (Spinreact company), Spain in 5% ice-cold 50 mM phosphate buffer 2- Uric acid:- pH 7.2. The homogenates were then Uric acid level was calculated centrifuged at 12000 × g for 12 min. spectrophotometrically using the The supernatantthus obtained was then commercial test of URIC ACID-LQ carefully collected and incubated with with spin lab (Spinreact company), 0.01 ml of absolute ethanol at 4°C for Spain.

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. The diagnosis of higher or lower value <0.01 was considered highly values of the previous parameters significant (HS) in all analyses. depends on on the mean value plus or minus standared deviation of rats of RESULTS control group. : 1- Biochemical study: B) Histological staining 1- As regard the control groups: Autopsy samples taken from the Group I (-ve control), group II (+ve liver and kidney of rats in different control group), and group III (vitamin groupswere processed according to C treated group). There was a non- Lamberg and Rothstein (1978), significant difference (p > 0.05) stained with Haematoxylin and Eosin between groups all over the period of (H&E), and examined at pathology the study by ANOVA test as regard: department in Benha faculty of 1- ALT, AST and hepatic MDA medicine. (table 1). C) Electron microscope study: 2- SOD, GPX, and catalase activity Autopsy samples taken from the in hepatic tissue (table 1). liver and kidney of rats in different 3- Uric acid, creatinine, and renal groups were processed according to MDA (tables 2). Maunsbach and Afzelius (1999), and 4- SOD, GPX, and catalase activity examined by transmission electron in renal tissue (table 2). microscope at 80 kilo volts. So the negative control group was Photographs were taken, developed, chosen as a representative group to be examined and printed at electron compared with the results of the treated microscope unit in Tanta University. groups; [group IV (bisphenol A treated Statistical analysis group), group V (bisphenol A + high The clinical data were recorded on dose of vitamin C treated group), and a report form. These data were group VI (bisphenol A + low dose of tabulated and analyzed using the vitamin C treated group)]. computer program SPSS (Statistical 2- As regard treated group: package for social science) version 16 1) Comparison between studied to obtain: groups (I,IV,V,VI) regarding liver Descriptive data enzymes (AST, ALT):- Descriptive statistics were The present study showed a calculated for the data in the form of significant increase in ALT & AST mean and standard deviation  SD.for levels in group IV (BPA treated group) quantitative data. group V (BPA + high dose of vitamin Analytical statistics C treated group) & non significant In the statistical comparison increase in their levels in group VI between the different groups, the (BPA + LOW dose of vitamin C treated significance of difference was tested group, as compared to group I (-ve using ANOVA test (F value) that used control group). but there was a to compare mean of more than two reduction in AST & ALT levels in groups of quantitative data. A P value groups V, VI as compared to group IV <0.05 was considered statistically which was significant for AST only significant (S) while P >0.05 was (Table 3). considered statistically insignificant, P

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2) Comparison between the dose of vitamin C) treated group as studied groups (I,IV,V,VI) compared to group IV (BPA group) regarding renal functions:- (Table 3). The present study showed an 5) Comparison between the increase in uric acid & creatinine levels studied groups (I,IV,V,VI) in groups IV ,V & an increase in regarding kidney MDA :- creatinine with decreased uric acid The present study showed an concentrations in group VI as increased level of MDA production in compared to group I. but there was a kidney in groups IV, V & VI as significant reduction in their compared to group I. The increased concentrations in groups V & VI as MDA in grou (BPA +high dose of vit compared to group IV (BPA treated c) p V was statistically significant as group) (Table 3). compared to group IV. But, there was a 3) Comparison between the non significant reduction in its studied groups (I,IV,V,VI) regarding concentration in group VI (BPA + low liver MDA (a marker of lipid dose of vit c) as compared to group IV peroxidation):- (BPA treated group) (Table 3). The present study showed an 6) Comparison between the increased level of MDA production in studied groups (I,IV,V,VI) group IV (BPA treated group), group V regarding kidney antioxidants:- (BPA + high dose of vitamin C treated The present study showed that group), and group VI (BPA + low dose kidney CAT, SOD, and GPX of vitamin C treated group) as concentrations in groups IV, V & VI compared to group I (–ve control were significantly lower than group I. group), also, there was a significantly Furthermore, there were significant increased level of MDA production in reduction in their concentrations in group V (BPA + high dose of vitamin (BPA + high dose of vitamin C) treated C treated group) & group VI (BPA + group as compared to BPA treated low dose of vitamin C treated group) as group. But, also there was a non- compared to group IV (BPA treated significant increase in CAT, SOD, and group) (Table 3). GPX concentrations in group VI (BPA 4) Comparison between the + low dose of vitamin C) as compared studied groups (I,IV,V,VI) to group IV (BPA treated group) regarding liver antioxidants:- (Table 3). The present study showed that the 2 – Histopathological results liver CAT, SOD, and, GPX The Liver Tissue: In control concentrations in groups IV, V & VI groups & vitamin C treated group, were significantly lower than group. Light microscopic examination of Furthermore, there were significant sections from the liver of these groups reduction in their concentrations in showed the same structures, the normal (BPA + high dose of vitamin C) treated histological picture of the liver was group as compared to BPA treated recorded. The liver sections revealed group. But, there was a non significant normal hepatic plates radiating from a reduction in CAT & a non significant thin walled central vein (arrow1) increase in SOD and GPX separated by blood sinusoids and concentrations in group VI (BPA + low polyhedral hepatocytes contained

Egypt J. Forensic Sci. Appli. Toxicol Vol 16 (2) December suppl 2016 Haroun et al. 63 rounded vesicular nuclei (Fig. 1). In epithelium with luminal dilatation was BPA treated group, disarrangement of detected as shown in (Fig. 9). In hepatic plates, dilatation and groups treated with BPA + vitamin C congestion of blood sinusoids and in high & low doses, the same congested dilated central vein as shown histopathological disruptions were seen in (Fig. 2), swollen hepatocytes with in both groups. Degeneration of renal severe vacuolar (hydropic) tubular epithelium with luminal degeneration as shown in (Fig. 3), dilatation, and atrophied renal Moreover, marked inflammatory corpuscles are shown in (Fig. 10, 11, cellular infiltrations were noticed in the 12 respectively). portal area as shown in (Fig. 4). In 3-Electron microscopic results BPA + vitamin C treated groups in The Liver Tissue: In control high & low doses, the same groups & vitamin C treated group, histopathological disruption were seen the results revealed normal hepatocytes in both groups of BPA + vitamin C in ultrastructure including mitochondria, high & low doses. There was a rough endoplasmic reticulum, and hydropic degeneration of hepatocytes, nucleus as shown in (fig. 13), while dilated blood sinusoids and BPA treated group, showed inflammatory cell infiltration in mitochondrial swelling as shown in interstitial tissue and in portal tracts as (Fig. 14), and lipid accumulation in the shown in (Fig. 5, 6, 7 respectively). liver cells as shown in(Fig. 15). Also, The Renal Tissue: In control rats treated with BPA and vitamin C groups & vitamin C treated group, in high & low doses showed light microscopic examination of mitochondrial swelling and lipid sections from the kidney showed accumulation in the liver cells as shown normal histological picture of renal in (Fig. 16, 17). corpuscles and tubules were recorded. The Renal Tissue: Control The renal corpuscle contained tuft of groups & vitamin C treated group, blood capillaries, glomerulus, there was normal podocytes with surrounded by two layers of Bowman's normal foot processes as shown in (Fig. capsule that separated by urinary space. 18). While BPA treated group showed The renal tubules consisted of proximal enlargement of foot processes of convoluteed tubules lined with large podocytes, and thickening of basement pyramidal cells with apical brush membrane of Bowman's capsule as borders, while the distal convoluted shown in (Fig. 19). Renal tissue in rats tubules lined with simple cuboidal cells treated with BPA and vitamin C in (Fig. 8). The bisphenol A treated high & low doses revealed also group showed degenerative changes in enlargement of foot processes of the renal corpuscles and tubules, podocytesas shown in (Fig. 20, 21). atrophied renal corpuscles with Figures for the negative control group contracted glomerular tufts and were representative for other control widening of the urinary space. group & vitamin C treated group. Degeneration of renal tubular

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Table (1): Comparison between studied groups [group 1 (-ve control group), group II (+ve control group treated with the vehicle), and group III (treated with vitamin C 60 mg/kg)] regarding hepatic parameters (ALT, AST, MDA. CAT, SOD & GPX) Group I (-ve Group II (+ve Group III (Vit ANOVA P Control group) Control group) C ttt group) value Mean ± SD Mean ± SD Mean ± SD ALT 83.12±11.24 78.1±10.78 78.76±11.71 0.60 0.55 AST 151.96±12.0 152.88±13.67 147.09±10.71 0.66 0.52 MDA 52.74±17.92 39.19±11.74 44.93±17.3 1.94 0.16 CAT 76.62±4.71 75.2±9.72 71.99±8.57 0.88 0.43 SOD 52.53±8.36 54.28±7.38 56.64±4.20 0.90 0.42 GPX 32.92±4.26 33.08±3.77 31.97±4.05 0.23 0.80

Table (2): Comparison between studied groups [group 1 (-ve control group), group II (+ve control group treated with the vehicle), and group III (treated with vitamin C 60 mg/kg)] regarding renal parameters (creatinine, uric acid, MDA, CAT, GPX & SOD). Group I (-ve Group II (+ve Group III (Vit P Control group) Control group) C ttt group) ANOVA value Mean ± SD Mean ± SD Mean ± SD Creatinine 0.598±0.35 0.663±0.12 0.752±0.27 0.89 0.42 Uric acid 1.72±0.21 1.87±0.24 1.81±0.30 0.96 0.40 MDA 56.54±15.96 44.53±11.51 47.19±13.74 2.16 0.14 CAT 65.09±4.59 53.44±22.4€ 59.2±10.82 1.59 0.22 GPX 28.78±2.42 30.02±2.16 30.35±2.3 1.32 0.28 SOD 56.63±5.43 52.0±12.01 55.43±11.83 0.58 0.57

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Table (3) Comparison between the studied groups regarding liver enzymes, renal functions, and oxidative stress parameters Group I Group IV Group V Group VI ANO P (-ve (group ttt (group ttt with (group ttt VA value Control with BPA) BPA+high with group) Mean ± SD dose vit C) BPA+low Mean ± Mean ± SD dose vit C) SD Mean ± SD ALT 83.12±11 103.67±23.79 96.0±15.65∞^ 92.25±23.19 3.61 0.007 .24 €∞^ ** AST 151.96±1 213.8±17.24€ 187.96±43.06 162.31±32.27 11.45 0.001 2.0 ∞^ €∞^$ $# ** MDA 52.74±17 62.76±23.74 133.85±36.36 66.6±46.31$# 13.2 0.001 .92 ∞ €∞^$ ** CAT 76.62±4. 51.76±9.29€ 33.79±10.49 49.68±9.85€ 34.77 0.001 71 ∞^ €∞^$ ∞^# ** SOD 52.53±8. 33.25±9.63€ 16.08±5.71€∞ 34.31±10.23€ 37.07 0.001 36 ∞^ ^$ ∞^# ** GPX 32.92±4. 22.93±4.09€ 14.89±5.29€∞ 28.95±11.94$ 11.57 0.001 26 ∞^ ^$ # ** Creatin 0.598±0. 0.968±0.23€ 0.761±0.135 0.66±0.18$ 3.01 0.019 ine 35 ∞ * Uric 1.72±0.2 2.53±0.56€∞ 1.93±0.35$ 1.29±0.75$ 7.66 0.001 acid 1 ^ ** MDA 56.54±15 69.5±27.11∞ 97.8±33.66€∞ 68.12±23.8∞ 7.29 0.001 .96 ^ ^$ ^# ** CAT 65.09±4. 30.31±8.24€ 33.0±15.02€∞ 31.66±6.33€ 13.82 0.001 59 ∞^ ^ ∞^ ** GPX 28.78±2. 21.77±5.16€ 12.36±5.67€∞ 24.16±6.56∞ 22.31 0.001 42 ∞^ ^$ $# ** SOD 56.63±5. 27.45±8.3€∞ 15.29±7.39€∞ 30.24±7.3€∞ 33.11 0.001 43 ^ ^$ ^# ** €=Sig against group I ∞=Sig against group II ^=sig &group III $=sig & group IV #=sig & group V *= Sig **=highly significant

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Figure (1): A photomicrograph of a section in rat’s liver prepared from control group showing normal hepatic plates radiating from a thin walled central vein (arrow1) separated by blood sinusoids (black arrows), polyhedral hepatocytes contained rounded vesicular nuclei (white arrows) (H&E, x 40).

Figure (2):A photomicrograph of a section in rat’s liver prepared from BPA treated group showing congested dilated central vein (star) and dilated blood sinusoids (arrow) (H&E, x 20).

Figure (3): A photomicrograph of a section in rat’s liver prepared from BPA treated group showing hydropic degeneration of hepatocytes(arrows)(H&E, x 40).

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Figure (4): A photomicrograph of a section in rat’s liver prepared from BPA treated group showing inflammatory cell infiltration of portal tract (H&E, x 40).

Figure (5): A photomicrograph of a section in rat’s liver prepared from rat treated with BPA + vitamin C in high dose showing hydropic degeneration of hepatocytes (white arrows)& dilated blood sinusoids (black arrows)(H&E, x 40).

Figure (6): A photomicrograph of a section in rat’s liver treated with BPA + high dose of vitamin C showing mononuclear cell infiltration of portal tract (black arrow)), dilated blood sinusoids(white arrows)(H&E, x 40).

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Figure (7): A photomicrograph of a section in rat’s liver prepared from bisphenol A + low dose of vitamin C treated group showing dilated central vein (CV) and dilated blood sinusoids (arrows) (H&E, x 400).

Figure (8): A photomicrograph of a section in rat’s liver prepared from bisphenol A + low dose of vitamin C treated group showing mononuclear cell infiltration in portal tract (black arrow) and hydropic degeneration of hepatocytes (white arrows) (H&E, x 400).

Figure (9): A photomicrograph of kidney section of rat from controlgroup showing normal histological structure: renal corpuscles contain normal glomerular tuft (GT) & normal urinary space (U), proximal convoluted tubules (black arrows), and, distal convoluted tubules (white arrows)(H&E, x 40).

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Figure (10): A photomicrograph of kidney section of treated rat with bisphenol A (group B) showing atrophied renal corpuscles (white arrow) with contracted glomerular tuft (GT) and wide urinary space (U), and atrophy of renal tubular epithelium with luminal dilatation (black arrows)(H&E, x40).

Figure (11): A photomicrograph of a kidney section of treated rat with BPA + high dose of vitamin C showing atrophied renal corpuscles with contracted glomerular tuft (GT) and wide urinary space (U), and degeneration of renal tubular epithelium with luminal dilatation (RT) (H&E, x400).

Figure (12): A photomicrograph of a kidney section of treated rat with BPA + low dose of vitamin C showing atrophied renal corpuscles with contracted glomerular tuft (GT) and wide urinary space (U), and degeneration of renal tubular epithelium with luminal dilatation (RT) (H&E, x400).

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Figure (13): Electron micrograph of rat liver from control group showing normal nucleus. Normal mitochondria (black arrows),normal rough endoplasmic reticulum (arrow 1), and normal bile canaliculus (arrow 2) (TEM x3000).

Figure (14): Electron micrograph of rat liver treated with bisphenol A showing mitochondrial swelling (arrows) (TEM x3000).

Figure (15): Electron micrograph of rat liver treated with bisphenol A showing accumulation of lipid droplets (arrows) & swollen mitochondria (arrow 1) (TEM x3000),

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Figure (16): Electron micrograph of rat liver treated with BPA + vitamin C in high dose showing lipid droplet and mitochondrial swelling (arrows)(TEM x3000).

Figure (17): Electron micrograph of rat liver treated with bisphenol A + vitamin C in high dose showing accumulation of lipid droplets (arrows) (TEM x1500)

Figure (18): An electron photomicrograph of a section of rat liver treated with bisphenol A + low dose of vitamin C showing accumulation of lipid droplets (black arrow) & mitochondrial swelling (white arrows) (TEM x3000)

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Figure (19): Electron micrograph of rat kidney from control group showing normal foot processes of podocytes (black arrows) & normal thickness of basement membrane (white arrows) (TEM x5000)

Figure (20): Electron micrograph taken from kidney of rat treated with bisphenol A showing enlarged foot processes of podocytes (black arrows) & thickening of basement membrane (white arrows) (TEM x5000)

Figure (21): Electron micrograph taken from kidney of rat treated with bisphenol A and vitamin C showing enlarged foot processes of podocytes (arrows)(TEM x2000).

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Figure (22): An electron photomicrograph taken from kidney of rat treated with bisphenol + low dose of vitamin C showing enlarged foot processes of podocytes (black arrows) & thickening of basement membrane (white arrow) (TEM x5000).

DISCUSSION administration of BPA and vitamin C Bisphenol-A has been had a protective effect on BPA-induced demonstrated in both in vivo and in toxicity on the epididymis. At the vitro experiments to act as an endocrine beginning of the study, we disrupting chemical released into the hypothesized that vitamin C could act environment (Richter et al., 2007). as an antioxidant against BPA induced The majority of reported BPA toxicity oxidative damage in rats. studies in literature have focused on The present study revealed that reproductive effects of these chemicals BPA induced hepatotoxicity as there (Aydogan and Barlas, 2006). Besides were significant increase in aspartate its inherent effects on endocrine aminotransferase (AST), and alanine system, BPA is also known to inflict aminotransferase (ALT) over control oxidative stress by affecting the redox values in BPA treated group, status in the exposed organs significant increase in MDA which is a (Hasselberg et al., 2004). marker of lipid peroxidation (one of the Vitamin C is a very powerful chief mechanisms of cell damage) in antioxidant agent. One major function BPA treated group against control of vitamin C is to protect tissues from group, and the present study also harmful oxidation products (Korkmaz revealed significant (p<0.05) reduction and Kolankaya, 2009). However, in activities of enzymatic antioxidants there are limited data concerning the such as superoxide dismutase (SOD), effect of vitamin C on endocrine catalase (CAT), and glutathione disrupting chemical (EDC) induced peroxidase (GPX) in BPA treated oxidative stress in tissues. Chitra et al. group as compared to control. Data (2003) have suggested that co- from the present study demonstrated that bisphenol-A induced oxidative Egypt J. Forensic Sci. Appli. Toxicol Vol 16 (2) December suppl 2016 Haroun et al. 74 stress by reducing antioxidative enzymes, (superoxide dismutase, capacity of the liver. catalase, glutathione reductase and The present results are in glutathione peroxidase) in agreement with those of Korkmaz et mitochondrial and microsome-rich al. (2010) who reported a significant fractions of liver. increase in ALT and AST levels in rats The results of the present study are treated with 25 mg/kg BPA for 50 also in agreement with the study of days. These results are also in Hassan et al.,(2012) who found accordance with the study of Neha et reduction in both hepatic reduced al., (2012) who found a significant glutathione (GSH) and glutathione increase in AST and ALT levels in peroxidase (GSHPX) levels in rats mice treated with BPA (50 mg/kg) for treated with BPA (25 mg/kg/day) for 30 days. The study of Walaa et al., 30 days. As the increase in GSH level (2015), revealed that the activities of is important for GSHPX, which serum ALT and AST exhibited a requires GSH as a cofactor, and the significant increase in BPA treated elevation in GSH level increases group compared to control group. activity of GSHPX. The results of the current study are The results of the present study in accordance with the findings of contradicted with Bindhumol et al., previous study which had demonstrated (2003), who found that the activity of the involvement of oxidative stress and alanine transaminase (ALT) remained lipid peroxidation in BPA exposure had unchanged in BPA treated rats as induced liver toxicity (Lam et al., compared with the corresponding 2011).In line with this, another study control rats.AST and ALT enzymes are reported that BPA induced damage was the most reliable markers of associated with oxidative stress hepatocellular injury or necrosis. Their (Hassan et al., 2012).The study done levels are elevated in a variety of by Korkmaz et al. (2010), showed hepatic disorders. Of the two, ALT is significant increase in MDA level in thought to be more specific for hepatic the liver tissues of rats of BPA treated injury because it is present mainly in group as compared to control group. liver cystosol and in low concentration The results of the current study are elsewhere (Giboney, 2005). When the also in agreement with Asahi et al., liver hepatocytes are damaged, these (2010) who revealed that BPA enzymes are released into the blood increased the generation of reactive where the significant increase in AST oxygen species (ROS) and induced and ALT levels indicates the damage to cellular apoptosis in hepatocytes. the cystosol and also to mitochondria Moon et al., (2012) indicated that BPA (Mathuria and Verma, 2008). can induce hepatic damage and Regarding the histopathology of mitochondrial dysfunction by the liver in BPA treated group, the increasing oxidative stress in the liver. present study revealed changes in The results of the present study are hepatic architecture in the form of in agreement with the study of vacuolation and hydropic degeneration Bindhumol et al. (2003)who found of hepatocytes. Similar observations that bisphenol A generated ROS by were recorded by previous studies of decreasing the activities of antioxidant Hassan et al., (2012), Hussein and

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Eid, (2013), and Roy et al., (2011). In which has been implicated in the addition, dilated congested central vein, pathogenesis of hepatic steatosis. dilated blood sinusoids, and, The present study revealed that mononuclear cell infiltration in portal BPA induced nephrotoxicity by tract and in interstitial tissue were significant increase in detected. These findings agreed with malondialdehyde(MDA) which is a the results recorded by Daniela-Saveta marker of lipid peroxidation (one of the et al., (2014). chief mechanisms of cell damage) in The histopathological changes in BPA treated group against control the liver may be explained the ability of group, significant (p<0.05) reduction in BPA to induce apoptosis and cell activities of enzymatic antioxidants damage by the induction of adenylate such as superoxide dismutase (SOD), kinase activation, TNF-alpha gene catalase (CAT), and glutathione expression, dysregulation of Ca+2 peroxidase (GPX) in bisphenol A homeostasis, and production of ROS treated group as compared to control, (Reactive Oxygen Species) (Lee et al., also there were significant increase in 2008 and Kovacic, 2010). uric acid &creatinine levels in BPA Regarding electron microscopic treated group as compared to control study of the liver in BPA treated group, group. the current study revealed The results of the present study are mitochondrial swelling, and lipid in agreement with the study of accumulation in the liver cells. Korkmaz et al., (2009), that revealed The results of the present study are significant increase in MDA levels in in accordance with the study of Wei et renal tissue of rats treated with BPA al., (2014) who found that BPA (50mg/kg) for 50 days as compared to induced mitochondrial swelling during control group. Similarly, some previous in vitro study when mitochondria works have been demonstrated that isolated from neonatal rat liver were BPA generates ROS that caused treated with BPA at 30°C for 55 oxidative damage in the brain, minutes, reproductive tract and kidney of rats BPA acts directly on mitochondria, (Aydogan et al., 2008 and Aydogan et altering mitochondrial ultrastructure, al., 2009). inducing permeability transition (PT) The results of the present study are and releasing proteins that lead to also in agreement with the study of activation of apoptosis. In addition, the Korkmaz et al., (2009), that revealed release of apoptogenic proteins from significant reduction in GSH- based the mitochondria induced by BPA was antioxidant enzymes system including further demonstrated to cause glutathione peroxidase (GPX),and condensed chromatin in isolated liver glutathione reductase (GR) in renal nuclei (Snyder et al., 2000). tissues in BPA treated group as As regard lipid accumulation in the compared to control group. liver cells, this result agreed with Huc There are similarities between the et al., (2012)who made invitro study renal function alteration detected in the and reported that BPA-induced lipid present study and those described by accumulation in HepG2 cells by previous authors who found increase in disturbing mitochondrial function creatinine and Blood Urea Nitrogen

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(BUN) levels due to BPA activity of catalase enzyme had been exposure(Yildiz&Barlas, 2013 and detected in rat renal tissue after the Daniela-Saveta et al., 2014). daily oral administration of BPA at the In agreement with the results of the two dose levels (5, 10 mg/kg). The only current study, Murmu and significant changeobtained was an Shrivastava, (2014), showed that BPA increase in Glutathione-S-Transferase insignificantly increased the creatinine (GST) activity after 6 weeks of oral level after 15, 30 and 60 days administration of 25 mg/kg of BPA administration in fresh water fish (Mourad, &Khadrawy, 2012). (Cirrhinusmrigala) as compared to Regarding the histopathology of control. the kidney in rats treated with BPA, the Bisphenol A has a nephrotoxic present study revealed atrophy of renal effect due to accumulation of BPA corpuscles with retraction of toxic metabolites and inability of the glomerular tuft, widening of urinary kidney to eliminate those (Sangai et space, with degenerative changes of al., 2012). Several toxic substances that renal tubules, and mononuclear cell induced nephrotoxicity may result in infiltration. These results are in alteration of glomerular basement agreement with the study of Walaa et membrane and affecting glomerular al., (2015) and Korkmaz et al., filtration rate (Manikkam et al., 2013). (2009). The results of the present study are This may be explained by the also in agreement with the study of ability of BPA to produce reactive Mourad and Khadrawy, (2012) who oxygen species causing oxidative found a significant increase in uric acid damage and may lead to mitochondrial concentration occurred after 6 weeks of injury consequently playing an the daily oral administration of BPA at important role in apoptotic mechanisms both the high (25 mg/kg) and low (10 (Al-shobaili et al., 2013). mg/kg) dose levels. Regarding electron microscopic It has been demonstrated that study of the kidney in BPA treated serum uric acid levels are significantly group, the present study revealed elevated in non-alcoholic fatty liver enlargement of foot processes of patients (Li et al., 2009). Marmugi et podocytes, and thickening of basement al. (2011) suggested that low doses of membrane of Bowman's capsule. BPA may influence de novo fatty acid The results of the present study are synthesis thereby contributing to in accordance with the study of Olea- hepatic steatosis. Furthermore, uric Herreroet al., (2014) who found that acid increase may be due to the effect intraperitoneal injection of bisphenolA of BPA on the heart as several studies in mice at dose of 50 mg/Kg for 5 showed an association between weeks induced an enlargement of both elevated uric acid levels cytoplasm and foot processes of andcardiovascular diseases (Fang and podocytes as well as the presence of Alderman, 2000). condensed chromatin, suggesting On contrast to the results of the apoptosis. This is in line with the study present study, non-significant changes of Walaa et al., (2015) which revealed in lipid peroxidation and reduced that BPA induced thickening of the glutathione (GSH) levels and in the basement membrane of Bowman’s

Egypt J. Forensic Sci. Appli. Toxicol Vol 16 (2) December suppl 2016 Haroun et al. 77 capsule which confirmed by strong glutathione peroxidase (GSHPX) Periodic acid Schiff (PAS) stainability. catalyze dismutation of the superoxide Several toxic substances induced anion (O-2) into hydrogen peroxide nephrotoxicity may result in alteration (H2O2) which then convert hydrogen of the glomerular basement membrane peroxide to water, providing protection and affecting the glomerular filtration against reactive oxygen species (Manikkam et al., 2013). (Sayed-Ahmed et al., 2010).Therefore, BPA was able to induce podocyte the damage at the cellular level by hypertrophy by its effect on the oxidants is attenuated by antioxidant podocyte expression of the cycline- enzymes such as SOD, GSHPX, and dependent kinase inhibitor p27Kip1, CAT (Koc et al., 2005). which is known to play a key role in The significantly reduced activities the mechanism of renal cell of antioxidant enzymes might be due to hypertrophy beside its effect on BPA induced protein oxidation. The Transforming Growth Factor-β1 (TGF- interaction of lipid peroxidation (LPO) β1) system (Ziyadeh & Wolf, 2008 products with enzyme molecules leads and Romero et al., 2010). to the exclusive modification of The findings of the present study histidine residue and generation of clearly indicated the involvement of protein-protein cross linked derivatives oxidative stress caused by generation of causing reduction in enzyme activity reactive oxygen species in bisphenol-A (Kwon et al., 2000). induced hepatotoxicity and The results of the present study nephrotoxicity. Bisphenol A is revealed reduction in AST, ALT, and lipophilic in nature, so it can easily non significant improvement in MDA penetrate and interact with the lipid in rats treated with bisphenol A and content of cell membrane. Oral vitamin C in low dose as compared to administration of bisphenol A for 6 BPA treated group and -ve control weeks has resulted in significant group. There was also non significant elevation in MDA levels which could improvement in antioxidant enzymes be due to overproduction of reactive (CAT, SOD, and GPX) in the liver in oxygen species and suppression of the same group as compared to antioxidant enzymes levels resulting in bisphenol A treated group and –ve the altered redox potential of cell control group. Also, there were causing lipid peroxidation and cell reduction in AST, ALT, and significant damage. Several studies reported the reduction in MDA in rats treated with occurrence of oxidative toxicity after bisphenol A and vitamin C in high dose bisphenol-A exposure in rats and mice as compared to BPA treated group and (Doerge & Fisher, 2010 and Gong -ve control group. There was and Han, 2006). significant reduction in antioxidant Enzymatic antioxidants include enzymes (CAT, SOD, and GPX) in the superoxide dismutase (SOD), catalase, liver in the same group as compared to glutathione peroxidase (GPX), and bisphenol-A treated group and –ve glutathione-s-transferase (GST), which control group. constitutes the first line defense against The results of the present study are reactive oxygen species (ROS) induced in accordance with the study of damage (Droge, 2002). Catalase and Korkmaz et al., (2010)s who found

Egypt J. Forensic Sci. Appli. Toxicol Vol 16 (2) December suppl 2016 Haroun et al. 78 that serum ALT and AST levels were mitochondrial swelling, and lipid significantly higher (P < 0.05) in BPA accumulation in the liver cells. and BPA + vitamin C, treated groups In the kidney, the results of the when compared to control group of present study revealed a significant rats. In addition, serum ALT levels in increase in MDA level in renal tissues the BPA + vitamin C treated group in BPA + high dose of vitamin C were significantly higher compared to treated group as compared to BPA BPA treated group (P < 0.05). Also treated group and control group. On the they found that malondialdehyde other hand, there was a non significant (MDA) levels were significantly higher increase in MDA production in kidney in BPA + vitamin C treated group when in group VI (BPA + low dose of compared to BPA treated group and vitamin C treated group) as compared control group (P < 0.05). On the other to group I (-ve control group) & a non hand, there was significant reduction in significant reduction in its the liver catalase (CAT), superoxide concentration in this group as dismutase (SOD), and glutathione compared to group IV (BPA treated peroxidase (GPX) in BPA + vitamin C group). Regarding renal antioxidants, treated group as compared to BPA the results of the present study revealed treated group and control group. a significant reduction in renal CAT, The results of the present study GPX, and SOD concentrations in group contradicts with the study of V treated with (BPA + high dose of Bindhumol et al., (2003) who found vitamin C) as compared to group I (-ve that co-administration of vitamin C control group) & There was a reversed the effect of BPA induced significant reduction in GPX & SOD oxidative stress in the liver of rats. and a non significant increase in CAT Regarding the histopathology of concentrations in this group as the liver in rats treated with BPA + compared to group IV (BPA treated vitamin C in high & low doses, there group). While in group VI (BPA + low were more hydropic degeneration of dose of vitamin C treated group), there hepatocytes, dilated blood sinusoids was a significant reduction in renal and, marked inflammatory cell CAT, GPX, and SOD concentrations as infiltration in interstitial tissue and in compared to group I (-ve control portal tracts. group), also there was a non-significant These results are in accordance increase in CAT, SOD, and GPX with the study of Korkmaz et al., concentrations in this group as (2010) who revealed that vitamin C co- compared to group IV (BPA treated administration with BPA resulted in group). more severe hepatic congestion, more These results are in accordance necrotic areas, and mononuclear cell with the study of Korkmaz et al., infiltration when compared to BPA (2009) who found that treated group, malondialdehyde (MDA) levels in the Regarding electron microscopic renal tissue were found to be higher in study of the liver in rats treated with the BPA and BPA+ vitamin C treated BPA + vitamin C , in high & low doses groups compared with controls the current study revealed (P<0.05). Furthermore, higher MDA levels were detected in the BPA+

Egypt J. Forensic Sci. Appli. Toxicol Vol 16 (2) December suppl 2016 Haroun et al. 79 vitamin C treated group compared to CONCLUSION the BPA treated group. - Oral administration of The results of the present study bisphenol-A in a dose of 25 mg / kg didn't agree with the study of Korkmaz produced hepatotoxicity and et al., (2009), who found that serum nephrotoxicity by affecting creatinine level in the BPA + vitamin C oxidant/antioxidant balance in rat liver treated group were significantly lower and kidney that proved by biochemical compared with BPA treated group, and & histopathological changes. that vitamin C co-administration with - Co-administration of vitamin C BPA did not produce significant in low dose (5.5 mg/kg) didn't prevent increase in Blood Urea Nitrogen this oxidative damage. While co- (BUN) & uric acid concentration as administration of vitamin C in high compared to BPA treated group. dose (60 mg/kg) augmented this Regarding the histopathology of oxidative damage. the kidney in rats treated with BPA + vitamin C in high & low doses, the RECOMMENDATION same histopathological changes of the  Establishing a national policy kidney in rats treated with BPA were that restrict bisphenol-A use and detected, including degenerated renal promote alternative approaches tubules and atrophied renal corpuscles. respecting health and environment. These results are in accordance  Widespread public education with the study of Korkmaz et al., regarding the health hazards of BPA (2009) who revealed that necrotic with a special concern about its lesions, congestion areas, and systemic toxic effects, and encouraging mononuclear cell infiltration in the rat the use of alternative methods for kidneys of BPA + vitamin C treated hardness of polycarbonate plastics and group were similar to those observed in epoxy resins. BPA treated group.  Use glass, or polypropylene Regarding electron microscopic bottles instead of polycarbonate ones study of the kidney in in rats treated (hard, shiny,clear or tinted plastic, with BPA + vitamin C in high & low usually with a number 7 or “PC” on the doses, the present study revealed bottom). enlargement of foot processes of  To clean polycarbonate bottles, podocytes similar to that of rats treated do not use harsh detergents or put with BPA. bottlesin the dishwasher. Instead, clean The unpredicted damaging effect of them with warm soapy water and a vitamin C in this study may be sponge. Scouring brushes can scratch explained by prooxidant effect, usually the surface of the bottles and increase by interaction with transition metal leaching rates. ions. When Fe+3is present, vitamin C  Avoid heating foods in can convert Fe+3 into Fe+2 which reacts polycarbonate containers, as bisphenol with oxygen or hydrogen peroxide A tends to leach faster with higher resulting in formation of hydroxyl temperatures. Use glass or ceramic radicals and superoxide anions containers instead. (Rietjens et al., 2002).

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 Avoid use of infant formula Immunological Investigation, 42, brands incans that use BPA as an epoxy 191-203. liner. Aydogan, M. and Barlas, N. (2006):  Cut back on consumption of Effects of maternal 4-tert- canned foodsand beverages to reduce octylphenol exposure on the exposure to bisphenolA contamination reproductive tract of male rats at from the interiorcoating of the adulthood. Reprod. Toxicol. 22, container. Also, avoid cannedfoods 455–460. with higher fat content, which mayhave Aydogan, M., Korkmaz, A. , Barlas, higher levels of bisphenol A. N. and Kolankaya, D. (2008): The  Avoid use of high dose of effect of vitamin C on bisphenol A, vitamin C as antioxidant with exposure nonylphenol and octylphenol to BPA. induced brain damages of male  Other studies are required to rats. Toxicology, 249: 35-39. evaluate the toxic effects of BPA and Aydogan, M., Korkmaz, A., Barlas, the results of co–administration of N. and Kolankaya, D. (2009): Pro vitamin C. oxidant effect of vitamin C  More studies are needed on coadministration with bisphenol A, vitamin C antioxidant and pro-oxidant nonylphenol, and octylphenol on properties. the reproductive tract of male rats. Drug Chem. Toxicol.. ACKNOWLEDGMENT Bindhumol, V., Chitra, K.C. and Our deep gratitude and thanks to all Mathur,. PP. (2003): Bisphenol A staff members in Electron Microscopic induces reactive oxygen species Unit, Faculty of Medicine, Tanta generation in the liver of male University, for their great help and rats.Toxicology 188:117–124. cooperation. Our great thanks to all of Carr, A. and Frei, B. (1999:Does the staff members in Pathology Vitamin C act as a pro-oxidant Department, Faculty of Medicine, under physiological conditions? Benha University for their great help to FASEB J;13:1007–24. finish this work. Lastly we would like Chattopadhyay, A., Biswas, S., to extend our thanks to all of the staff Bandyopadhyay, D., Sarkar, C. members in Forensic Medicine & and Datta, A.G. (2003): Effect of Clinical Toxicology Department, isoproterenol on lipid peroxidation Faculty of Medicine, Benha University, and antioxidant enzymes of for their help & cooperation. myocardial tissue of mice and www.fmed.bu.edu.eg protection by quinidine. Molecular and Cellular Biochemistry 245, 43- REFERENCES 49. Chitra, K.C., Rao, K.R. and Mathur, Al-Shobaili,H., Al-Robaee,A., P.P. (2003): Effect of bisphenol A Alzolibani, and Rasheed, Z. and coadministration of bisphenol (2013): Immunological studies of A and vitamin C on epididymis of reactive oxygen species damaged adult rats: a histopathological and catalase in patients with systemic biochemical study. Asian J. lupus erythmatosis correlation with Androl. 5, 203–208. disease activity index. Egypt J. Forensic Sci. Appli. Toxicol Vol 16 (2) December suppl 2016 Haroun et al. 81

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a,H.M.,Cnubbenb, N.H.P.,van tetrachlorobisphenol A and Zandena,J.J.,vander,W.H., pentachlorophenol on the Alinka,G.M. and Koemana JH. transcriptional activities of (2002): the pro-oxidant chemistry androgen receptor-mediated of the natural antioxidants vitamin reporter gene. Food Chem C, vitamin E, carotenoids and Toxicol;44(11):1916-21. flavonoids. Environ Tian, X., Takamoto, M., Sugane, K. ToxicolPharmacol 11:321–333. (2003): Bisphenol A promotes IL-4 Romero, M., Ortega, A., Izquierdo, production by Th2 cells. Int Arch A., Lopez-Luna, P. and Bosch, Allergy Immunol;132(3):240-247. R.J. (2010): Parathyroid hormone- Vandenberg, L.N., Chahoud, I., related protein induces hypertrophy Heindel, J.J.,Padmanabhan, V., in podocytes via TGF-beta(1) and Paumgartten, F.J. p27(Kip1): Implications for andSchoenfelder, G.(2010). diabetic nephropathy. Nephrol Dial Urinary, circulating, and tissue Transplant 25:2447–2457. biomonitoring studies indicate Roy, S., Kalita C.J.andMazumdar, widespread exposure to bisphenol M. (2011): Histopathlogical effects A. Environ. Health Perspect., 118: of Bisphenol A on liver of 1055-1070. HeteropneustesFossilis (Bloch). An Walaa, M.S., Ahmed, Walaa, A., international Quarterly Journal of Moselhy and Nabil, T.M. (2015): Environmental Sciences Bisphenol A Toxicity in Adult theEcoscan, 1: 187-190. Male Rats: Sangai, N.P., Verma R.J. andTrivedi, Hematological,Biochemical and M.H. (2012):Testing the efficacy Histopathological Approach. of quercetin in mitigating bisphenol Global Veterinaria,14 (2): 228-238. A toxicity in liver and kidney of Wei, X., Ying J., Yuanyuan, L.i., mice. Toxicology and Industrial Yanjian, W., Juan, L., Yue, M., Health, pp: 1-17. Zhenxing, M., Huailong, C., Sayed-Ahmed, M. M., Aleisa, A. M. Gengqi, L., Bing, X., Xi, C. and and Al-Rejaie , S. S. (2010): Shunqing, X. (2014): Early-Life Thymoquinone attenuates Exposure to Bisphenol A Induces diethylnitrosamine induction Liver Injury in Rats Involvement of ofhepatic carcinogenesis through Mitochondria Mediated Apoptosis. antioxidant signaling,” Oxidative PLoS One. 9(2): e90443. Medicine and Cellular Longevity, Yildiz, N. and Barlas, N. (2013): vol. 3, no. 4, pp. 254–261. Hepatic and renal functions in Snyder, R.W., Maness, S. C., Gaido , growing male rats after bisphenol K.W., Welsch ,F. and Sumner, A and octylphenol exposure. S.C. (2000): Metabolism and Human & Experimental disposition of bisphenol A in Toxicology, 32: 675-686. female rats. ToxicolApplPharmacol Ziyadeh, F.N. and Wolf, G. (2008). 168: 225–234. Pathogenesis of the podocytopathy Sun, H., Xu, L.C., Chen, J.F., Song, and proteinuria in diabetic L. and Wang, X.R. (2006):Effect glomerulopathy.Curr Diabetes Rev of bisphenol A, 4:39 45.

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تأثير فيتبييٍ ج عهي ضًيخ يبدح انجطفينىل أ عهي انكجذ و انكهي في فئراٌ انتجبرة انجيضبء

د/ يبرضيم ريطيص هبرو1ٌ, د/ إثراهيى ضيذ زيــــسو1,د/ إضالو ضبيى يتىني1 , رثبة شعجبٌ انشبفعى2. 1 قطى انطت انشرعى و انطًىو األكهينيكيخ, كهيخ انطت, جبيعخ ثنهب, يصر, 2 يذرش يطبعذ ثقطى انطت انشرعى و انطًىو األكهينيكيخ, كهيخ انطت, جبيعخ ثنهب انًهخــــــــــص انعرثــــــــــى اىبسفْٞ٘ه أ ٕ٘ ٍبدة ٍسببت إلخخاله اىغذد اىصَبء ٍٗسخخذٍت فٜ جَٞع أّحبء اىعبىٌ فٜ اىعذٝذ ٍِ اىصْبعبث اىبالسخٞنٞت، ٗحعشض اىبشش ىٖزٓ اىَ٘اد ٝبذأ فٜ ٗقج ٍبنش خاله اىحٞبة اىجْْٞٞت. ٗقذ أثبسث اىعذٝذ ٍِ اى٘مبالث ححزٝشاث ظذ االسخخذاً اىَفشغ ىٖزٓ اىَبدة. ٕذفج اىذساست اىحبىٞت إىٚ حقٌٞٞ حأثٞش االمسذة اىْبحجت عِ ٍبدة اىبسفْٞ٘ه أ عيٚ اىنبذ ٗاىنيٚ فٜ اىفئشاُ اىبٞعبء، ٗ عَب إرا مبُ إظبفت فٞخبٍِٞ ج َٝنِ أُ ٝخفف ٕزا اىعشس اىخأمسذٛ. حٌ حقسٌٞ اىفئشاُ اىبٞعبء إىٚ ثالد ٍجَ٘عبث: اىَجَ٘عت اىعببطت، اىَجَ٘عت اىخٜ ع٘ىجج ببىبسفْٞ٘ه أ، ٗ اىَجَ٘عت اىخٜ ع٘ىجج ببىبسفْٞ٘ه أ + فٞخبٍِٞ ج. حٌ حقٌٞٞ االمسذة اىْبجَت عِ اىبسفْٞ٘ه أ فٜ أّسجت اىنبذ ٗاىنيٚ ، ٗببإلظبفت إىٚ رىل، حٌ قٞبط ٍسخٛ٘ اىنشٝبحِْٞٞ ٗ حَط اىٞ٘سٝل فٜ اىذً، ٗ ّشبغ إّضَٝبث اىنبذ معالٍبث ى٘ظبئف اىنبذ ٗ اىنيٚ. مشفخبىخحبىٞو اىنَٞٞبئٞت اىحٝ٘ٞت عِ اّخفبض مبٞش فٜ أّشطت ٍعبداث األمسذة األّضَٞٝت فٜ اىَجَ٘عت اىخٜ ع٘ىجج ببىبسفْٞ٘ه أ، ٗ اىَجَ٘عت اىخٜ ع٘ىجج ببىبسفْٞ٘ه أ + فٞخبٍِٞ ج ٍقبسّت ببىَجَ٘عت اىعببطت. ٗببإلظبفت إىٚ رىل، مبّج ْٕبك صٝبدة مبٞشة فٜ ٍسخٝ٘بث إّضَٝبث اىنبذ، ٗحَط اىٞ٘سٝل، ٗ اىنشٝبحِْٞٞ فٜ اىَجَ٘عت اىخٜ ع٘ىجج ببىبسفْٞ٘ه أ، ٗ اىَجَ٘عت اىخٜ ع٘ىجج ببىبسفْٞ٘ه أ + فٞخبٍِٞ ج ٍقبسّت ببىَجَ٘عت اىعببطت. ٗقذ ى٘حظج حغٞشاث ّسٞجٞت ٍشظٞت فٜ اىنبذ ٗاىنيٚ فٜ اىَجَ٘عت اىخٜ ع٘ىجج ببىبسفْٞ٘ه أ، ٗ اىَجَ٘عت اىخٜ ع٘ىجج ببىبسفْٞ٘ه أ + فٞخبٍِٞ ج ٍقبسّت ببىَجَ٘عت اىعببطت. أظٖشث اىذساست اىحبىٞت أُ اىبسفْٞ٘ه أ ىٔ حأثٞش سَٜ عيٜ اىنبذ ٗاىنيٜ بفئشاُ اىخجبسة اىبٞعبء، عِ غشٝق اىخأثٞش عيٚ اىخ٘اصُ بِٞ اىَ٘اد اىَؤمسذة ٍٗعبداث ىألمسذة،ٗأُ إظبفت فٞخبٍِٞ ج ٍع اىبسفْٞ٘ه أ أدٙ إىٜ حقٝ٘ت ٕزا اىعشساىخأمسذٛ.

Egypt J. Forensic Sci. Appli. Toxicol Vol 16 (2) December suppl 2016