Studies on the potential protective effect of cinnamon against bisphenol A- and octylphenol-induced oxidative stress in male albino rats

Among the numerous chemicals discharged into the surrounding environment, bisphenol A (BPA) and octylphenol (OP) have been shown to increase oxidative stress in body by disturbing the prooxidant/antioxidant balance of cells. Cinnamon aqueous extract (CAE) is a natural product rich in polyphenolic compounds that have antioxidant activity. This study was designed to investigate the protective efficacy of CAE against oxidative disorders induced by BPA and OP in male albino rats. Animals were divided into 6 groups (10 rats each) and treated orally, 3 times weekly for 50 days. Group 1: control vehicle (olive oil); group 2 (25 mg BPA/kg b.wt./day); group 3 (25 mg OP/kg b.wt./day); group 4 (200 mg CAE/kg b.wt./day); group 5 (CAE 2 h before BPA administration); and group 6 (CAE 2 h before OP administration). BPA- and OP-exposed groups showed insignificant elevation in the final body weight; weight gains and significant reduction only in the relative kidneys weight. Also, BPA and OP exposure resulted in significant increase in serum urea, creatinine and kidney, brain, testicular malondialdehyde (MDA) levels. Significant reduction in tissues reduced glutathione (GSH) contents; catalase (CAT) and superoxide dismutase (SOD) activities were also recorded in BPA and OP exposed animals compared to the control vehicle group. Pretreatment with CAE 2 h either before BPA or OP administration ameliorated the BPA- and OP-induced body weight; weight gains and relative organs weight changes and biochemical adverse effects. CAE pretreatment also protected against the recorded pathological changes in kidney, brain and testis. In conclusion, CAE could ameliorate the oxidative toxic effects of BPA and OP indicating its protective antioxidant effect.


Introduction
Environmental pollution with hazardous chemicals and metals can arise from natural as well as anthropogenic Bisphenol A (BPA) and octylphenol (OP) are endocrine disrupting chemicals (EDCs) that are of scientific interest due to their widespread use and ubiquitous exposures. Beside their endocrine disrupting effect [28,34,56] they are also known to induce oxidative stress [2,3,25,26].
BPA is the building block of polycarbonate plastics, a hard plastic used to make numerous consumer products, including most baby and water bottles. Its final product includes adhesives, coatings, paints and building materials [57]. BPA waste may enter the environment during handling, loading and unloading, heating or releases. BPA can enter the human body through reusable baby bottles [8], food packing materials [27], liquid of canned vegetables [10] and dental sealants [41].
OP is one of the final degradation products of alkylphenol polyethoxylates (APEOs), which are nonionic surfactants used as intermediates or as additives for a wide range of industrial products and processes [19,47]. OPs are used as tackifiers in tire rubber, in recovery of oil in offshore processes, and in printing inks, pesticide formulations (as a dispersant), water based paints, textile auxiliaries, and emulsion polymerization processes [37]. These are generally discharged in large quantities to aquatic environments either directly from untreated effluent or indirectly from sewage treatment plants [33]. Exposure to OPs may occur through ingestion of contaminated foods (e.g. fish) and drinking contaminated water. It may occur also from contact with some personal care products and detergents [12].
Generation of free radicals and antioxidant capacity of the body have been observed to be modulated by environmental, physiological and nutritional factors [31,44]. For example, physiological factors such as aging, alteration of body mass index and obesity and life style confounding factors such as smoking, drinking and high calorie diet have enhancing effect on oxidative stress and suppressive effect on antioxidants [44,48,49].
Natural plant-derived antioxidants are in high demand than synthetic antioxidants because of their potential in health promotion, disease prevention, and their improved safety and consumer acceptability [20,54].
Cinnamon is one of the most widely used plants in herbal medicines with diverse bioactive effects. It is obtained from the inner bark of several trees from the genus Cinnamomum. The used species in this study, Cinnamomum aromaticum (C. cassia or Chinese cinnamon) is an ever green tall tree with thick leathery leaves and yellow flowers [35].
Su et al. [58] indicated that cinnamon may serve as potential dietary source of natural antioxidants for improving human nutrition and health. It is rich in natural polyphenolic compound. Polyphenols act as reactive oxygen and nitrogen species scavengers, redox-active transition metal chelators and enzyme modulators [45].
Previous studies reported that cinnamon stimulates the increase of antioxidant enzymes activities, including SOD and CAT in rat's liver and heart [15,24].
The present study was designed to assess the potential protective effect of Cinnamon cassia aqueous extract against bisphenol A-and octylphenol-induced oxidative stress in the kidney, brain and testis of male albino rats.

Animals
A total of 60 adult male albino rats weighing between 120 and 150 g were used in this study. They were obtained from the AL-Zyade Experimental Animals Production Center, Giza, Egypt. All animals were housed in polypropylene cages with mesh wire tops in well ventilated room and provided with balanced ration and clean water ad libitum. They were kept under observation for two weeks before the beginning of experiments for atmospheric and handling accommodation.
C. cassia bark was purchased from Harraz market, EL-Azhar, Cairo, Egypt. It was identified and authenticated in Botany Department, Faculty of Science, Cairo University.
Diagnostic kits for assaying serum urea and creatinine levels; MDA, GSH levels and CAT, SOD activities in renal, brain and testicular tissue homogenates were purchased from the Biodiagnostic Company, Dokki, Giza, Egypt.

Preparation of cinnamon aqueous extract (CAE)
The C. cassia aqueous extract (CAE) was prepared from the air dried powdered cinnamon bark according to Azab et al. [4]. The aqueous extract was freshly prepared by soaking 10 g of the grinded bark in 100 ml distilled water at 90 • C for 2 h followed by filtration. The filtrate was then dehydrated in oven at 80 • C overnight. The resulting dark reddish brown dry extract was weighed and the dry yield was then calculated.

Experimental design and animal grouping
Sixty male albino rats were weighed and divided into six equal groups; all were treated orally three times a week for 50 days [25,26].

Collection of serum and tissue samples
At the end of the experiment, the animals were fasted overnight and weighed then anaesthetized and sacrificed for samples collection. Blood samples were collected without anticoagulant and centrifuged at 3000 rpm for 15 min for serum separation. Table 1 Effects of BPA and OP exposure alone and 2 h after CAE administration on the final body weight (g); weight gains (g) and relative organ weights (%) of control and treated rats. Values are presented as mean ± S.E. (n = 10 animals/group). a Significantly different from group 1 at P < 0.05.
Kidneys, brain and testes of each rat were collected and divided into two parts, one part stored at −20 • C for tissue biochemical investigations while the other part was kept in 10% formalin for the histopathological examination.

Estimation of kidney functions
Serum urea and creatinine levels were measured using the colorimetric method of Fawcett and Soctt [18] and Schirmeister et al. [50], respectively.

Estimation of tissues lipid peroxidation product, MDA levels
MDA was determined in kidneys, brain and testes tissue homogenates according to the procedure described by Ohkawa et al. [39].

Estimation of tissues antioxidant parameters
GSH concentration, CAT and SOD activities were determined in Kidneys, brain and testes tissue homogenates according to Beutler et al. [7], Aebi [1] and Nishikimi et al. [38], respectively.

Histopathological examination
Kidney, brain and testes tissue samples intended for the histopathological investigations were fixed in 10% neutral formalin, prepared and stained according to Bancroft et al. [5].

Statistical analysis
Values are presented as mean ± standard error (SE). Statistical significance of toxic oxidative effects of BPA and OP on kidneys, brain and testes and the protective effect of CAE were determined by one way ANOVA according to Snedecor and Cochron [55]. All statistical analyses were performed using SPSS (Statistical package for Social Sciences) Version 16 released on 2007.

Effects on the final body weight, weight gain and relative organ weights
Oral administration of BPA (group 2) or OP (group 3) induced insignificant elevation in the final body weight; weight gains with significant reduction in the relative kidneys weight. However, brain and testes relative weights were insignificantly reduced compared to the control vehicle group (group 1). Oral administration of CAE 2 h before BPA (group 5) and OP (group 6) exposure induced insignificant decrease in the final body weight; weight gains compared to the BPA-(group 2) and OP-(group 3) exposed groups, respectively. However, the relative kidneys, brain and testes weights were insignificantly increased as revealed in Table 1.

Effects on kidney function (serum urea and creatinine levels)
The recorded nephrotoxic alterations in Table 2 revealed that oral administration of BPA (group 2) or OP (group 3) induced significant elevation in serum urea and creatinine levels compared to control vehicle group. CAE pretreatment 2 h before BPA (group 5) or OP (group 6) administration ameliorated their nephrotoxic effects as indicated by significant reduction in serum urea and creatinine levels compared to the BPA-(group 2) and OP-(group 3) treated groups, respectively.

Effects on the tissues lipid peroxidation product, MDA
The obtained results (Table 3) revealed that kidney, brain and testes MDA levels were significantly increased in BPA-(group 2) and OP-(group 3) treated groups compared to the control vehicle group. Oral administration of CAE 2 h before BPA or OP exposure, induced significant decrease in MDA levels in these organs compared to the  BPA-and OP-treated groups, respectively and became as that of control.

Effects on tissues antioxidant defense system
Significant reduction in kidney, brain and testicular GSH concentrations, CAT and SOD activities of BPA-(group 2) and OP-(group 3) exposed animals was observed on comparison with the control vehicle group. CAE pretreatment 2 h before BPA (group 5) or OP (group 6) administration ameliorated these effects and kept kidney, brain and testicular GSH concentrations and CAT, SOD activities within normal range compared to control vehicle group (Tables 4 and 5).
In all the investigated parameters, CAE alone-treated group showed insignificant changes compared to the control one.

Effects on histological structures of the investigated organs
BPA and OP oral administration alone at 25 mg/kg/day (three times/week for 50 days each) induced histopathological changes in the kidneys, brain and testis of exposed rats. However, CAE pretreatment (200 mg/kg/day, orally, three times/week for 50 days) 2 h before BPA and OP exposure ameliorated these alterations by variable degrees (Figs. 1-3).  Fig. 1.
Kidney of olive oil-treated rats (group 1) showed normal renal glomeruli and tubules with slight congestion. The kidney sections of BPA-exposed rats (group 2) showed glomerular congestion and hypercellularity; cloudy swelling in proximal convoluted tubules (PCT) with obstructed lumen and intertubular congestion. Similarly, the kidney of OP-exposed rats (group 3) showed glomerular congestion, hemorrhage and hypercellularity, cloudy swelling in proximal convoluted and collecting renal tubules.
Kidney of CAE-treated rats (group 4) showed more or less normal renal glomeruli and tubules with slight congestion.
Pretreatment with CAE 2 h before BPA (group 5) or OP (group 6) intoxication ameliorated most of the observed changes in renal morphology. This was represented by slight glomerular congestion and hypercellularity with more or less normal renal tubules in the CAE + BPAtreated group. The same protective effect of CAE was observed in CAE + OP-treated rats but without hypercellularity.

Table 5
Effects of BPA and OP exposure alone and 2 h after CAE administration on CAT and SOD activities (U/g tissue) in kidney, brain and testicular tissues of control and treated rats.

Enzyme
CAT SOD Values are presented as mean ± S.E. (n = 10 animals/group). a Significantly different from group 1 at P < 0.05. b Significantly different from group 2 at P < 0.05. c Significantly different from group 3 at P < 0.05.

3.2.4.2.
Effects on the brain. BPA-and OP-induced brain oxidative damage and the protective effect of CAE pretreatment 2 h prior their administration are shown in Fig. 2. Normal cerebellum and white matter were observed in the brain of the olive oil-exposed animals. The obtained photomicrographs from brain of BPA-exposed rats showed central chromatolysis in neuron, pyknosis, perineuronal satellitosis and neuronophagia in mid brain (motor area). Brain sections of OP-exposed rats showed chromatolysis, pyknosis, perineuronal microgliosis and neuronophagia.
CAE-treated rat's brain showed more or less normal cerebellum and white matter.
Pretreatment with CAE 2 h before BPA or OP intoxication protected against BPA-and OP-induced brain morphological alterations that was indicated by the recorded more or less normal brain cerebellum and white matter of the CAE + BPA-treated animals and of the CAE + OP-treated ones.

3.2.4.3.
Effects on the testes. The observed testicular histopathological alterations induced by BPA and OP exposure alone and the ameliorating effect of CAE pretreatment 2 h prior their administration are shown in Fig. 3.
Photomicrograph of testis of olive oil-treated rats showed more or less normal seminiferous tubules.  However, testes of BPA-treated rats showed hyperemia, perivascular edema, testicular degeneration and cellular debris in the lumen. Also, testis of OP-treated rats showed hyperemia, perivascular edema and testicular degeneration.
CAE + BPA-treated rats showed more or less normal seminiferous tubules. Similarly, normal seminiferous tubules were also seen in CAE + OP-treated animals.

Discussion
Exposure to several chemicals and environmental contaminants has been reported to increase oxidative stress in body by disturbing the prooxidant/antioxidant balance of cells [2,22]. BPA and OP were previously reported to induce oxidative damage in several tissues [2,3,25,26].
It is widely accepted that antioxidants may delay or inhibit the oxidation of lipids or other molecules by inhibiting the initiation, or propagation of oxidizing chain reactions [61]. These involve absorption and neutralization of free radicals, quenching singlet and triplet oxygen, and decomposing peroxides [42].
Considerable attention has recently been focused on some nutritional factors such as polyphenols that could counteract oxidative damages and therefore be beneficial through their antioxidant properties [32,45]. Among plants that contain polyphenols is cinnamon that may be of special interest, since cinnamon and CAE has been documented to be in vitro and in vivo antioxidant [4,30] and [17].
In consistent with our results, various investigators reported that BPA or OP exposure of rats and mice resulted in insignificant difference in final b.wt., weight gain, relative brain and testes weights [2,9,23,25,59,[62][63][64][65] but significant decrease in relative kidneys weight was observed [66]. On the contrary, significant changes in body weight, weight gain and relative organs weight were reported by others [46,59,[67][68][69]. These discrepancies in weight changes may be related to the differences in doses, routes, duration, and time of exposure; rat and mice strains, sex and nature of food.
The recorded insignificant increase in body weight could be attributed to the ability of BPA to stimulate the accumulation of triacylglycerol in 3T3-L1 preadipocytes [70,71]; to affect gene expression in fatty tissues [72] and genes involved in metabolic syndrome [73] and to hypothyrodism [74] that resulted in weight gain, enhancement of glucose transport in adipocytes [75].
Several animal studies showed that over-stimulation of the estrogen receptor-alpha (ER␣) in pancreatic ␤-cells by estrogen or BPA produced an excessive insulin signaling in the liver, endothelium and in fat, thus potentially leading to obesity, glucose intolerance, and dyslipedimia [76][77][78][79].
Bisphenol A and OP administration has been reported to cause an increase in the thiobarbituric acid reactive substances (TBARS) levels in the kidneys, brain and testes of male rats [2,3,26]. Kabuto et al. [80] attributed the underdevelopment of the kidneys, brain, and testes to the increased levels of TBARS in BPA-exposed mice. This may explain the recorded decrease in relative kidneys, brain and testes weights.
The reduction in body weights with CAE pretreatment may be resulted from its ability to decrease fat mass percent [81]. Also, the increased relative kidneys, brain and testes weights may be referred to its antioxidant properties and the reduction of TBARS levels in these organs [4,15].
In coincidence with our results, Suberg et al. [82] and Saleh [83] recorded increased serum urea and creatinine levels after BPA or OP oral administration. On contrary, Til et al. [66] recorded decreased creatinine levels in male rats at and above 500 mg/kg BPA.
The elevated urea and creatinine levels recorded in our results may be resulted from the observed BPA-and OP-induced peroxidative effects that were confirmed by the photographed renal histopathological alterations. Also, BPAand OP have been reported to induce hyperglycemia that may result in kidney damage and renal dysfunction and consequently greatly increased serum urea, creatinine and uric acid [84,85].
The nephroprotective effects of cinnamon were previously recorded by El-Yamani [85] who observed decreased serum urea and creatinine levels in diabetic rats treated with cinnamon.
In the present study, BPA and OP administration resulted in lipid peroxidation in kidney, brain and testicular tissues that was represented by the significantly increased MDA levels and reduced GSH concentrations and CAT; SOD activities. However, CAE pre-treatment ameliorated these oxidative effects.
BPA and OP exposure caused oxidative stress by disturbing the balance between ROS and antioxidant defenses system in kidney, brain and testis of rats [2,3,26,86] and mice administered BPA throughout the embryonic/fetal life and during infancy [80].
BPA has been shown to be decomposed to many kinds of metabolites including BPA radical by a reaction with radical oxygen [87]. Several evidences indicated that BPA exerts potent oxidant activity in various tissues via ROS production which results in inhibition of antioxidant enzymes; increased H 2 O 2 and lipid peroxidation products, LPO [9,13,14,23,88].
The present study confirmed the previously recorded antioxidant effect of cinnamon in vitro [52] and in vivo [17] that was indicated by decreased MDA levels and increased antioxidant enzymes activities. Murcia et al. [36] found that cinnamon extract exhibited protective capacity against irradiation-induced LPO in liposomes, and quenched hydroxyl radicals (OH • ) and H 2 O 2 . The protective action of cinnamon extract against radiation-induced oxidative and inflammatory damages was attributed to its suppressive effect on ROS generation. This is due to its phenolic and flavonoids contents, in addition to modification of gene expression by inhibiting nuclear factor-kappa B (NF-Kb) activation [4]. Polyphenols act as reactive oxygen and nitrogen species scavengers, redoxactive transition metal chelators and enzyme modulators [45]. The high reactivity of the hydroxyl substituent of flavonoids with the number of hydroxyl groups on the B-ring being correlates with ROS scavenging capability [11,21,51].

Conclusion
In conclusion, the results of this study revealed that oral BPA and OP administration induced adverse oxidative effects on the exposed animals as evidenced by the recorded abnormalities in the investigated biochemical parameters. In addition, there were histopathological alterations in the investigated organs. Pretreatment with CAE provided a protective antioxidant role against such adverse effects.