Article
Neuroprotective effect of quercetin against oxidative damage and neuronal apoptosis caused by cadmium in hippocampus
Toxicology and Industrial Health 1–10 © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233713504810 tih.sagepub.com
Mehmet Kanter1, Cuneyt Unsal2, Cevat Aktas3 and Mustafa Erboga3 Abstract The purpose of the present investigation was to evaluate cadmium (Cd)-induced neurotoxicity in hippocampal tissues and beneficial effect of quercetin (QE) against neuronal damage. A total of 30 male rats were divided into 3 groups: control, Cd-treated, and Cd þ QE-treated groups. After the treatment, the animals were killed and hippocampal tissues were removed for biochemical and histopathological investigation. Cd significantly increased tissue malondialdehyde (MDA) and protein carbonyl (PC) levels and also decreased superoxide dismutase (SOD) and catalase (CAT) enzyme activities in hippocampal tissue compared with the control. Administration of QE with Cd significantly decreased the levels of MDA and PC and significantly elevated the levels of antioxidant enzymes in hippocampal tissue. In the Cd-treated group, the neurons of both tissues became extensively dark and degenerated with pyknotic nuclei. The morphology of neurons in Cd þ QE group was well protected, but not as neurons of the control group. The caspase-3 immunopositivity was increased in degenerating neurons of the Cd-treated group. Treatment of QE markedly reduced the immunoreactivity of degenerating neurons. The results of the present study show that QE therapy causes morphologic improvement in neurodegeneration of hippocampus after Cd exposure in rats. Keywords Cadmium, Quercetin, hippocampus, oxidative stress, neuronal damage
Introduction The main threats to human health from toxic metals are associated with exposure to lead, cadmium (Cd), mercury, and arsenic. Cd is a ubiquitous environmental and industrial pollutant that accumulates in animals and humans (Waisberg et al., 2003). The sources of human exposure to Cd include primary metal industries, production of certain batteries, intake of contaminated food or water, and inhalation of tobacco smoke or polluted air (Lo´pez et al., 2006; Nawrot et al., 2006). The targets for Cd toxicity include the kidney, lung, gastrointestinal tract, bone, and also the central nervous system (CNS) (Lehman and Klaassen, 1986; Nordberg, 1984; Webster and Valois, 1981). Although it is difficult for foreign substances to cross the blood–brain barrier (BBB), after chronic low-level exposure, Cd may affect the integrity or permeability of the barrier and
reach the CNS (Murphy, 1997; Shukla et al., 1996). Numerous animal studies have demonstrated behavioral disorders, morphological, and biochemical changes in the CNS in Cd-exposed experimental animals (De Castro et al., 1996; Webster and Valois, 1981). In clinical and epidemiological studies,
1
Department of Histology and Embryology, Faculty of Medicine, Istanbul Medeniyet University, Istanbul, Turkey 2 Department of Psychiatry, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey 3 Department of Histology and Embryology, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey Corresponding author: Mehmet Kanter, Department of Histology and Embryology, Faculty of Medicine, Istanbul Medeniyet University, Istanbul 34710, Turkey. Email: [email protected]
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cognitive function disabilities have also been observed in Cd-exposed populations (Marlowe et al., 1983; Viaene et al., 2000). Cd induces neurotoxicity with a wide spectrum of clinical entities including oxidative stress, changes in normal neurochemistry of the brain, memory impairment, cerebral hemorrhage, and edema (Gonc¸alves et al., 2010; Me´ndez-Armenta and Rı´os, 2007; Viaene et al., 2000). One of the effects induced by Cd is the enhancement of lipid peroxidation (LPO), which is dependent on free oxygen radicals. Cd penetrates the BBB and accumulates into the brain that is among the most susceptible organs to Cdinduced LPO (Gutierrez-Reyes et al., 1998; Kumar et al., 1996). The activity of antioxidant defense systems for preventing the damage to the tissues by oxygen radicals is diminished or inhibited in growing rats exposed to Cd (Shukla et al., 1996). Quercetin (QE), a flavonoid of natural origin, can readily cross the vascular endothelial barrier (Youdim et al., 2004). It is an efficient antioxidant (Arredondo et al., 2010), with proven neuroprotective activity (Ossola et al., 2009). QE might cross the BBB; its neuroprotective effects have been demonstrated in a variety of brain injury models showing improvement in morphological and functional outcomes (Cho et al., 2006). In in vitro study with PC12 cell line, QE showed inhibitory effect against cell damage (Gelinas and Martinoli, 2002). QE also attenuated neuronal damage following focal brain ischemia in in vivo model. Youdim et al. (2004) reported that QE can pass the BBB. Furthermore, QE has been known to reduce nitric oxide production by microglia (Kao et al., 2010). Hippocampus is a major component of the brains and belongs to the limbic system. It plays important roles in the consolidation of information from shortterm memory to long-term memory and spatial navigation. The hippocampus is located in the medial temporal lobe of the brain. In Alzheimer’s disease, the hippocampus is one of the first regions of the brain to suffer damage (Amaral and Lavenex, 2006). As the hippocampus is an important compartment in the memory formation, we examine the possible beneficial effects of QE on neurodegeneration in hippocampus after Cd exposure in rats.
Materials and methods Animals A total of 30 adult male Sprague–Dawley rats (3month-old), weighing 280–320 g, were used in this
study. Rats were provided by the Experimental Research Center of the Medical Faculty of Trakya University, Turkey. The rats were kept in a windowless animal quarter where temperature (21 + 1 C) and illumination were automatically controlled (light on at 07 a.m. and off at 09 p.m., i.e. 14-h light/10-h dark cycle). Humidity ranged from 50 to 55%. The Ethical Committee of Trakya University approved all animal procedures and the experimental protocol. All animals received proper care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals as prepared by the National Academy of Sciences and published by the National Institutes of Health, USA. QE was obtained from Sigma Chemical (St Louis, Missouri, USA) and dissolved in 0.5 ml of 20% ethanol just before intraperitoneal (i.p.) injection (15 mg/kg). The control group was injected with the same volume of saline as the Cd-treated groups.
Experimental design A total of 30 male Sprague–Dawley rats were divided into 3 groups: control, Cd-treated and Cd þ QEtreated groups; each group contained 10 animals. Control animals received daily injections of the saline vehicle alone. The Cd-treated group was injected subcutaneously with cadmium chloride dissolved in saline at a dose of 2 ml/kg/day for 30 days, resulting in a dosage of 1 mg/kg Cd. The rats in QE-treated groups were given i.p. injection of QE (15 mg/kg body weight) once a day starting 2 days prior to the Cd injection during the study period. At the end of the study, all animals were anesthetized with an i.p. injection of sodium thiopenthal (100 mg/kg, Sigma, St Louis, Missouri, USA). Twenty minutes later, the anesthetized rats were killed and immediately the hippocampal tissues were removed for biochemical and histopathological investigation.
Biochemical procedures Preparation of tissue samples. Tissue samples were frozen at 70 C and irrigated well with a solution of sodium chloride (0.9%). By admixing it with potassium chloride (1.5%), homogenization at a ratio of 1:10 was achieved. The DIAX9000 Homogenizer (Heidolph Instruments, Germany) was used to homogenize the tissue samples. MDA determination. The lipid peroxide level in the centrifuged tissue homogenates was measured according
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to the method described by Ohkawa et al. (1979). The reaction product was assayed spectrophotometrically (model UV-1700; Shimadzu, Kyoto, Japan) at 532 nm. The lipid peroxide level was expressed as the nanomole of malondialdehyde (MDA) per milligram of hippocampal tissue protein. Protein levels were measured according to the method described by Lowry et al. (1951). PC determination. Oxidative damage to proteins (protein carbonyl (PC)) was assessed by the determination of carbonyl groups based on the reaction with dinitrophenylhydrazine, as previously described (Levine et al., 1990). Briefly, proteins were precipitated by the addition of 20% trichloroacetic acid and redissolved in dinitrophenylhydrazine and the absorbance was read at 370 nm. The results were calculated using the extinction coefficient of 22,000 for aliphatic hydrazone. Results were expressed in nanomoles per milligram tissue. SOD determination. The superoxide dismutase (SOD) activity was determined according to the method described by Sun et al. (1988). The principle of the method is based on the inhibition of nitroblue tetrazolium reduction by the xanthine–xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the lyzate after 1.0 ml ethanol/chloroform mixture (5/3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the nitroblue tetrazolium reduction rate. SOD activity was also expressed in units per mg protein. CAT determination. Catalase (CAT) activity was determined according to Aebi’s method (1974). The principle of the method was based on the determination of the rate constant (s1, k) of the hydrogen peroxide (H2O2) decomposition rate at 240 nm. Results were expressed as k (rate constant) per milligram protein.
Histological examinations The brain tissue specimens were individually immersed in 10% neutral-buffered formalin dehydrated in alcohol and embedded in paraffin. Sections of 5 m were obtained, deparaffinized, and stained with hematoxylin and eosin (H&E). The brain tissue was examined and evaluated in random order under blindfold conditions with standard light microscopy.
Immunohistochemical procedures Harvested hippocampal brain tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 5 mm thickness. Immunohistochemical reactions were performed according to the avidin–biotin complex (ABC) technique described by Hsu et al. (1981). The procedure involved the following steps: (1) endogenous peroxidase activity was inhibited by 3% H2O2 in distilled water for 30 min; (2) the sections were washed in distilled water for 10 min; (3) nonspecific binding of antibodies was blocked by incubation with normal goat serum (DAKO X 0907, Carpinteria, California, USA) with phosphatebuffered saline (PBS), diluted 1:4; (4) the sections were incubated with specific rabbit polyclonal anticaspase 3 antibody (Cat. #RB-1197-P, Neomarkers, Fremont, California, USA), diluted 1:50 for 1 h at room temperature; (5) the sections were washed in PBS 3 3 min; (6) the sections were incubated with biotinylated anti-mouse immunoglobulin G (DAKO LSAB 2 Kit); (7) the sections were washed in PBS 3 3 min; (8) the sections were incubated with ABC (DAKO LSAB 2 Kit); (9) the sections were washed in PBS 3 3 min; (10) peroxidase was detected with an aminoethylcarbazole substrate kit (AEC kit; Zymed Laboratories, San Francisco, California, USA); (11) the sections were washed in tap water for 10 min and then dehydrated; (12) the nuclei were stained with hematoxylin; and (13) the sections were mounted in DAKO paramount. All dilutions and thorough washes between steps were performed using PBS unless otherwise specified. All steps were carried out at room temperature unless otherwise specified. Histological specimens of the hippocampal tissues were examined under light microscopy, with the examination carried out at a magnification of 400 and the counts of neurons determined per square millimeter with the use of a standardized ocular grid. Apoptotic cells (caspase-3 immunopositive) were counted. The distribution of neurons was examined in the sections from the specimens were subjected by immunohistochemical method (caspase 3 antibody). Tissue sections were examined under light microscopy (400), and the number of the apoptotic neurons counted within random high-power fields using a Nikon Optiphot 2 light microscope incorporating a square graticule in the eyepiece (eyepiece 10, objective 40, a total side length of 0.25 mm2).
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Table 1. Hippocampal tissue MDA and PC levels and SOD and CAT enzyme activities of all groups.a Control MDA (nm/mg tissue) PC (nm/mg tissue) SOD (U/mg protein) CAT (k/mg protein)
4.32 3.22 3.77 2.51
+ 0.44 + 0.24 + 0.29 + 0.19
Cadmium 5.99 2.29 2.80 1.72
+ + + +
0.54b 0.17b 0.22b 0.14b
Cadmium þ QE 5.18 2.76 3.45 2.09
+ 0.50c + 0.21c + 0.25c + 0.16c
MDA: malondialdehyde; PC: protein carbonyl; SOD: superoxide dismutase; CAT: catalase; QE: quercetin. a Kruskal–Wallis test was used for statistical analysis. Values are expressed as means + SD, n ¼ 10 for each group. b p < 0.01 compared with the control group. c p < 0.05 compared with control group.
Statistical analysis All statistical analyses were carried out using Statistical Package for Social Sciences software (SPSS for Windows, version 11.0; SPSS Inc., Chicago, Illinois, USA). All data were expressed as mean + SD. Differences in measured parameters among the three groups were analyzed with a nonparametric test (Kruskal–Wallis). Dual comparisons between groups exhibiting significant values were evaluated with Mann–Whitney U test. These differences were considered significant when p < 0.05.
Results Biochemical findings MDA levels of the tissues. MDA levels in the hippocampal tissue were found to be significantly higher in the Cd group than the control group. Treatment with QE prevented elevation of MDA levels significantly (Table 1). PC levels of the tissues. In the Cd group, there was a significant increase in protein oxidation levels in the hippocampal tissue, when compared with the control group. QE treatment significantly prevented protein oxidation (Table 1). SOD and CAT activities of the tissues. SOD and CAT activities in the hippocampal tissue was found to be significantly less in the Cd group than in the control group. The activities of both enzymes were kept at a level similar to the control group in Cd þ QE group (Table 1).
Histological findings In the control group, the morphology of neurons in CA2 region of the hippocampal tissue was normal
(Figure 1(a)). The most consistent findings in histologic sections of hippocampal neurons in Cd group stained with H&E were severe degenerative changes, shrunken cytoplasma, and extensively dark pyknotic nuclei (Figure 1(b)). Treatment with QE reduced the incidence of these degenerative changes in the hippocampal tissues and showed almost normal architecture similar to the control group (Figure 1(c)).
Immunohistochemical findings Light micrographs showed apoptotic hippocampal neurons after Cd exposure by caspase 3 immunohistochemistry (Figure 2). Mild caspase 3 immunoreactivity was observed in the cytoplasm of neurons in control rats CA2 region of the hippocampus (Figure 2(a)). The caspase 3 immunopositivity was increased in degenerating neurons in the CA2 region of the hippocampus following Cd exposure (Figure 2(b)). Treatment of QE markedly reduced the immunoreactivity of degenerating neurons after Cd exposure (Figure 2(c)). Moreover, the number of caspase 3 positive apoptotic neurons in the CA2 region of the hippocampus were increased significantly in Cd-treated rats compared with the control (p < 0.001) and Cd þ QE group (p < 0.01) rats’ hippocampal tissues (Table 2).
Discussion This experiment was carried out to evaluate the biochemical and histopathological changes in hippocampal tissues due to oxidative damage by Cd and protective effect of QE. Cd can disrupt the main structural components of the blood–brain and blood–cerebrospinal fluid barrier by injuring endothelial, epithelial, and glial cells and affecting the formation of tight junctions between barrier cells (Fleming et al., 2000; Virgintino et al.,
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Figure 1. Representative light microphotographs showing the histology of CA2 region of the hippocampus after Cd exposure. (a) In control group, normal hippocampal neurons are seen. (b) In Cd-treated rats, the severe degenerative changes in the cytoplasma and especially in the hippocampal cell nuclei of cells are seen. (c) Treatment with QE reduced the incidence of these degenerative changes and was less as compared to the Cd group (H&E, scale bar, 50 m). Cd: cadmium; QE: quercetin; H&E: hematoxylin and eosin.
2004). Then, Cd in circulation can enter the CNS and induce negative effects (Duval and Grubb, 1986). Cd has a long biological half-life (about 30 years in humans), which contributes to its accumulation in various tissues such as the brain (Usai et al., 1999). Cd is also toxic to neurons. The metal causes continuous elevation in intracellular calcium concentrations in neurons (Yoshida, 2001) and neuronal death by apoptosis or necrosis (Lopez et al., 2003). Cd has been reported to induce metallothionein expression in astrocytes (Rising et al., 1995). Cd can enter the body through inhalation, dermal exposure, and oral intake of contaminated food and drinking water. For example, electronic waste contains a number of toxic heavy metals, such as Cd, which can be released from ewastes through primitive recycling processes. Previously, Huo et al. (2007) reported that children living in e-waste dismantling site had significantly higher blood Cd levels as compared to those living in neighboring town. Brain aging is a risk factor for neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease. Oxidative damage plays a significant role in
brain aging (Johnson et al., 1999; Reiter, 1995), which induces overproduction of reactive oxygen species (ROS), decreases antioxidant enzyme activity and induces neuronal degeneration (Churchill et al., 2002; Colcombe et al., 2003). Oxidative stress represents an imbalance between the production of ROS including peroxides and free radicals, and the ability of biological system to readily detoxify the reactive intermediates or to repair the resulting damage (Sies, 1997). Disturbances in the normal redox state of tissues can cause toxic effects through the production of ROS that damage all components of the cell, including proteins, lipids, and DNA (Valko et al., 2007). Cells are protected against oxidative stress by an interacting network of antioxidant enzymes such as SOD and CAT (Sies, 1997). Many teratogenic mechanisms, including that of thalidomide have been linked to ROS production (Hansen et al., 2002). Cd has also been described as a potent inhibitor of antioxidant enzymes and closely associated with oxidative stress both in adult tissue (Shukla and Kumar, 2009) and in embryonic tissue (Paniagua-Castro et al., 2008). LPO is the process
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Figure 2. Representative light microphotographs showing apoptotic hippocampal neurons of CA2 region of the hippocampus after Cd exposure by caspase 3 immunohistochemistry. (a) In control rats, neuronal cells are moderately stained with the anticaspase 3 antibody. (b) In Cd-treated rats, the caspase 3 immunopositivity was strongly increased in CA2 neurons of the hippocampus after Cd exposure. (c) Treatment of QE markedly reduced the immunoreactivity of degenerating neurons after toluene exposure. (Immunoperoxidase, hematoxylin counterstain, scale bar, 50 m). Cd: cadmium; QE: quercetin. Table 2. The numbers (number per square micrometer) of apoptotic neurons (caspase 3 immunopositive) in the CA2 region of the hippocampal tissue of all groups.a Groups Control Cadmium Cadmium þ QE
Hippocampal tissue 6.2 + 0.8 31.1 + 4.6b 19.8 + 2.3c
QE: quercetin. a Kruskal-Wallis test was used for statistical analysis. Values are expressed as means + SD, n ¼ 10 for each group. b p < 0.001 compared with the control group. c p < 0.01 compared with control group.
of oxidative degradation of polyunsaturated fatty acids and its occurrence in biological membranes causes impaired membrane fluidity and inactivation of several membrane-bound enzymes (Goel et al., 2005). MDA is one of the major oxidation products of peroxidized polyunsaturated fatty acids, and thus increased MDA content is an important indicator of LPO (Demir et al., 2011). QE excerts its antioxidant action by scavenging free radicals, such as superoxide radicals generated by
xanthine/xanthine oxidase (Dok-Go et al., 2003). Additionally, QE exhibited peroxyl and hydroxyl freeradical scavenging activity (Kefalas et al., 2003). Free-radical scavenging effect of QE is due to its ability to chelate divalent cations. QE has the hepatoprotective (Renugadevi and Milton Prabu, 2009) and nephroprotective effect against the Cd-induced toxicity by increasing the methyl transferase and endothelial nitric oxide synthase expression (Morales et al., 2006). Additionally, a short time since it has been notified that QE can pass through the BBB of in situ models (2004). In addition, QE applies the protective effect in a stroke model induced by transient global ischemia (2006). QE significantly protected the neuronal cells from the oxidative stress-induced neurodegeneration in Alzheimer’s disease (Heo and Lee, 2004), decreased MDA level, improved activity of CAT and SOD (Mahesh and Menon, 2004). Furthermore, it was found that QE attenuated the neuronal death in the hippocampus resulting in improved learning and memory in arm maze test (Pu et al., 2007). Hence, these pieces of evidence point out the possibility that QE might exert an influence on the CNS.
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Therefore, we investigated the effects of QE on oxidative stress after Cd exposure. We show for the first time that Cd induced hippocampal tissue damage and apoptosis. In our current study, increases in MDA and PC levels, as well as decreases in SOD and CAT activity, tissue damage, and neuronal apoptosis induced by Cd detoxification were prevented by QE treatment, demonstrating that oxidative injury and apoptosis of the hippocampal tissue were attenuated by this treatment. To date, there is no information on the effect of QE on neuronal damage after Cd exposure in hippocampus. In a study, Mendez-Armenta et al. (2001) showed that there were degenerative changes that included pyknosis, necrosis, interstitial edema, and hyperchromatic cells in hippocampus of brain after Cd exposure. In another study, Gerspacher et al. (2009) reported that Cd exposure induces severe damage in primary cultures of both cortical neurons and glial cells, as demonstrated by vitality assays. Moreover, two different cell lines that we used as model of either neurons pheochromocytoma cells or brain capillary endothelial cells were also damaged by Cd. Our histological results show that Cd can cause diffuse neuronal damage. These results correlate well with the above literatures. The observed histological impairments by Cd have been recovered significantly by QE in QE þ Cd-treated rats indicating that QE is capable of preventing the neuronal damage induced by Cd. Therefore, it may be suggested that QE might inhibit Cd-induced hippocampal tissue damage. In other cellular systems, Cd produces either apoptotic or necrotic cell death depending on culture conditions as well as on the concentration and the duration of exposure. Apoptosis and necrosis are two morphologically and biochemically distinct processes of cell death, discernable based on cellular and organellar membrane integrity, nuclear fragmentation, enzymatic activation, and release of cellular content (Wyllie et al., 1980). Cd-induced apoptosis in vivo (Lag et al., 2002) and in vitro (Lasfer et al., 2008; Shih et al., 2003) was found to primarily involve the intrinsic mitochondrial-dependent pathway. Following chronic Cd exposure, there was an upregulation in gene expression of Bax (Fernandez et al., 2003; Shin et al., 2003), p53, p21, and a downregulation of Bcl-2 (Shin et al., 2003). Caspase 3, an effector caspase (Nicholson, 1999), has been shown to be critical in Cd-induced apoptosis in the above-mentioned studies. Kanter (2013) suggests that QE may be beneficial to neuronal apoptosis following Cd exposure in frontal cortex brain. In our current study, results supported
that the above-mentioned literatures reported on apoptosis in terms of neuronal survival. Moreover; the number of apoptotic neurons in hippocampal tissue of Cd-treated group was significantly more than both the control and Cd þ QE groups. We conclude that the natural flavonoid QE inhibits LPO, neuronal cell apoptosis, hippocampal neuronal cell damage, and raises antioxidant enzyme activity of the hippocampal tissue after Cd exposure. We believe that further preclinical research into the utility of QE may indicate its usefulness as a potential treatment on Cd-induced neurodegeneration. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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Neuroprotective effect of quercetin against oxidative damage and neuronal apoptosis caused by cadmium in hippocampus
Toxicology and Industrial Health 1–10 © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233713504810 tih.sagepub.com
Mehmet Kanter1, Cuneyt Unsal2, Cevat Aktas3 and Mustafa Erboga3 Abstract The purpose of the present investigation was to evaluate cadmium (Cd)-induced neurotoxicity in hippocampal tissues and beneficial effect of quercetin (QE) against neuronal damage. A total of 30 male rats were divided into 3 groups: control, Cd-treated, and Cd þ QE-treated groups. After the treatment, the animals were killed and hippocampal tissues were removed for biochemical and histopathological investigation. Cd significantly increased tissue malondialdehyde (MDA) and protein carbonyl (PC) levels and also decreased superoxide dismutase (SOD) and catalase (CAT) enzyme activities in hippocampal tissue compared with the control. Administration of QE with Cd significantly decreased the levels of MDA and PC and significantly elevated the levels of antioxidant enzymes in hippocampal tissue. In the Cd-treated group, the neurons of both tissues became extensively dark and degenerated with pyknotic nuclei. The morphology of neurons in Cd þ QE group was well protected, but not as neurons of the control group. The caspase-3 immunopositivity was increased in degenerating neurons of the Cd-treated group. Treatment of QE markedly reduced the immunoreactivity of degenerating neurons. The results of the present study show that QE therapy causes morphologic improvement in neurodegeneration of hippocampus after Cd exposure in rats. Keywords Cadmium, Quercetin, hippocampus, oxidative stress, neuronal damage
Introduction The main threats to human health from toxic metals are associated with exposure to lead, cadmium (Cd), mercury, and arsenic. Cd is a ubiquitous environmental and industrial pollutant that accumulates in animals and humans (Waisberg et al., 2003). The sources of human exposure to Cd include primary metal industries, production of certain batteries, intake of contaminated food or water, and inhalation of tobacco smoke or polluted air (Lo´pez et al., 2006; Nawrot et al., 2006). The targets for Cd toxicity include the kidney, lung, gastrointestinal tract, bone, and also the central nervous system (CNS) (Lehman and Klaassen, 1986; Nordberg, 1984; Webster and Valois, 1981). Although it is difficult for foreign substances to cross the blood–brain barrier (BBB), after chronic low-level exposure, Cd may affect the integrity or permeability of the barrier and
reach the CNS (Murphy, 1997; Shukla et al., 1996). Numerous animal studies have demonstrated behavioral disorders, morphological, and biochemical changes in the CNS in Cd-exposed experimental animals (De Castro et al., 1996; Webster and Valois, 1981). In clinical and epidemiological studies,
1
Department of Histology and Embryology, Faculty of Medicine, Istanbul Medeniyet University, Istanbul, Turkey 2 Department of Psychiatry, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey 3 Department of Histology and Embryology, Faculty of Medicine, Namik Kemal University, Tekirdag, Turkey Corresponding author: Mehmet Kanter, Department of Histology and Embryology, Faculty of Medicine, Istanbul Medeniyet University, Istanbul 34710, Turkey. Email: [email protected]
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cognitive function disabilities have also been observed in Cd-exposed populations (Marlowe et al., 1983; Viaene et al., 2000). Cd induces neurotoxicity with a wide spectrum of clinical entities including oxidative stress, changes in normal neurochemistry of the brain, memory impairment, cerebral hemorrhage, and edema (Gonc¸alves et al., 2010; Me´ndez-Armenta and Rı´os, 2007; Viaene et al., 2000). One of the effects induced by Cd is the enhancement of lipid peroxidation (LPO), which is dependent on free oxygen radicals. Cd penetrates the BBB and accumulates into the brain that is among the most susceptible organs to Cdinduced LPO (Gutierrez-Reyes et al., 1998; Kumar et al., 1996). The activity of antioxidant defense systems for preventing the damage to the tissues by oxygen radicals is diminished or inhibited in growing rats exposed to Cd (Shukla et al., 1996). Quercetin (QE), a flavonoid of natural origin, can readily cross the vascular endothelial barrier (Youdim et al., 2004). It is an efficient antioxidant (Arredondo et al., 2010), with proven neuroprotective activity (Ossola et al., 2009). QE might cross the BBB; its neuroprotective effects have been demonstrated in a variety of brain injury models showing improvement in morphological and functional outcomes (Cho et al., 2006). In in vitro study with PC12 cell line, QE showed inhibitory effect against cell damage (Gelinas and Martinoli, 2002). QE also attenuated neuronal damage following focal brain ischemia in in vivo model. Youdim et al. (2004) reported that QE can pass the BBB. Furthermore, QE has been known to reduce nitric oxide production by microglia (Kao et al., 2010). Hippocampus is a major component of the brains and belongs to the limbic system. It plays important roles in the consolidation of information from shortterm memory to long-term memory and spatial navigation. The hippocampus is located in the medial temporal lobe of the brain. In Alzheimer’s disease, the hippocampus is one of the first regions of the brain to suffer damage (Amaral and Lavenex, 2006). As the hippocampus is an important compartment in the memory formation, we examine the possible beneficial effects of QE on neurodegeneration in hippocampus after Cd exposure in rats.
Materials and methods Animals A total of 30 adult male Sprague–Dawley rats (3month-old), weighing 280–320 g, were used in this
study. Rats were provided by the Experimental Research Center of the Medical Faculty of Trakya University, Turkey. The rats were kept in a windowless animal quarter where temperature (21 + 1 C) and illumination were automatically controlled (light on at 07 a.m. and off at 09 p.m., i.e. 14-h light/10-h dark cycle). Humidity ranged from 50 to 55%. The Ethical Committee of Trakya University approved all animal procedures and the experimental protocol. All animals received proper care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals as prepared by the National Academy of Sciences and published by the National Institutes of Health, USA. QE was obtained from Sigma Chemical (St Louis, Missouri, USA) and dissolved in 0.5 ml of 20% ethanol just before intraperitoneal (i.p.) injection (15 mg/kg). The control group was injected with the same volume of saline as the Cd-treated groups.
Experimental design A total of 30 male Sprague–Dawley rats were divided into 3 groups: control, Cd-treated and Cd þ QEtreated groups; each group contained 10 animals. Control animals received daily injections of the saline vehicle alone. The Cd-treated group was injected subcutaneously with cadmium chloride dissolved in saline at a dose of 2 ml/kg/day for 30 days, resulting in a dosage of 1 mg/kg Cd. The rats in QE-treated groups were given i.p. injection of QE (15 mg/kg body weight) once a day starting 2 days prior to the Cd injection during the study period. At the end of the study, all animals were anesthetized with an i.p. injection of sodium thiopenthal (100 mg/kg, Sigma, St Louis, Missouri, USA). Twenty minutes later, the anesthetized rats were killed and immediately the hippocampal tissues were removed for biochemical and histopathological investigation.
Biochemical procedures Preparation of tissue samples. Tissue samples were frozen at 70 C and irrigated well with a solution of sodium chloride (0.9%). By admixing it with potassium chloride (1.5%), homogenization at a ratio of 1:10 was achieved. The DIAX9000 Homogenizer (Heidolph Instruments, Germany) was used to homogenize the tissue samples. MDA determination. The lipid peroxide level in the centrifuged tissue homogenates was measured according
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to the method described by Ohkawa et al. (1979). The reaction product was assayed spectrophotometrically (model UV-1700; Shimadzu, Kyoto, Japan) at 532 nm. The lipid peroxide level was expressed as the nanomole of malondialdehyde (MDA) per milligram of hippocampal tissue protein. Protein levels were measured according to the method described by Lowry et al. (1951). PC determination. Oxidative damage to proteins (protein carbonyl (PC)) was assessed by the determination of carbonyl groups based on the reaction with dinitrophenylhydrazine, as previously described (Levine et al., 1990). Briefly, proteins were precipitated by the addition of 20% trichloroacetic acid and redissolved in dinitrophenylhydrazine and the absorbance was read at 370 nm. The results were calculated using the extinction coefficient of 22,000 for aliphatic hydrazone. Results were expressed in nanomoles per milligram tissue. SOD determination. The superoxide dismutase (SOD) activity was determined according to the method described by Sun et al. (1988). The principle of the method is based on the inhibition of nitroblue tetrazolium reduction by the xanthine–xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the lyzate after 1.0 ml ethanol/chloroform mixture (5/3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the nitroblue tetrazolium reduction rate. SOD activity was also expressed in units per mg protein. CAT determination. Catalase (CAT) activity was determined according to Aebi’s method (1974). The principle of the method was based on the determination of the rate constant (s1, k) of the hydrogen peroxide (H2O2) decomposition rate at 240 nm. Results were expressed as k (rate constant) per milligram protein.
Histological examinations The brain tissue specimens were individually immersed in 10% neutral-buffered formalin dehydrated in alcohol and embedded in paraffin. Sections of 5 m were obtained, deparaffinized, and stained with hematoxylin and eosin (H&E). The brain tissue was examined and evaluated in random order under blindfold conditions with standard light microscopy.
Immunohistochemical procedures Harvested hippocampal brain tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 5 mm thickness. Immunohistochemical reactions were performed according to the avidin–biotin complex (ABC) technique described by Hsu et al. (1981). The procedure involved the following steps: (1) endogenous peroxidase activity was inhibited by 3% H2O2 in distilled water for 30 min; (2) the sections were washed in distilled water for 10 min; (3) nonspecific binding of antibodies was blocked by incubation with normal goat serum (DAKO X 0907, Carpinteria, California, USA) with phosphatebuffered saline (PBS), diluted 1:4; (4) the sections were incubated with specific rabbit polyclonal anticaspase 3 antibody (Cat. #RB-1197-P, Neomarkers, Fremont, California, USA), diluted 1:50 for 1 h at room temperature; (5) the sections were washed in PBS 3 3 min; (6) the sections were incubated with biotinylated anti-mouse immunoglobulin G (DAKO LSAB 2 Kit); (7) the sections were washed in PBS 3 3 min; (8) the sections were incubated with ABC (DAKO LSAB 2 Kit); (9) the sections were washed in PBS 3 3 min; (10) peroxidase was detected with an aminoethylcarbazole substrate kit (AEC kit; Zymed Laboratories, San Francisco, California, USA); (11) the sections were washed in tap water for 10 min and then dehydrated; (12) the nuclei were stained with hematoxylin; and (13) the sections were mounted in DAKO paramount. All dilutions and thorough washes between steps were performed using PBS unless otherwise specified. All steps were carried out at room temperature unless otherwise specified. Histological specimens of the hippocampal tissues were examined under light microscopy, with the examination carried out at a magnification of 400 and the counts of neurons determined per square millimeter with the use of a standardized ocular grid. Apoptotic cells (caspase-3 immunopositive) were counted. The distribution of neurons was examined in the sections from the specimens were subjected by immunohistochemical method (caspase 3 antibody). Tissue sections were examined under light microscopy (400), and the number of the apoptotic neurons counted within random high-power fields using a Nikon Optiphot 2 light microscope incorporating a square graticule in the eyepiece (eyepiece 10, objective 40, a total side length of 0.25 mm2).
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Table 1. Hippocampal tissue MDA and PC levels and SOD and CAT enzyme activities of all groups.a Control MDA (nm/mg tissue) PC (nm/mg tissue) SOD (U/mg protein) CAT (k/mg protein)
4.32 3.22 3.77 2.51
+ 0.44 + 0.24 + 0.29 + 0.19
Cadmium 5.99 2.29 2.80 1.72
+ + + +
0.54b 0.17b 0.22b 0.14b
Cadmium þ QE 5.18 2.76 3.45 2.09
+ 0.50c + 0.21c + 0.25c + 0.16c
MDA: malondialdehyde; PC: protein carbonyl; SOD: superoxide dismutase; CAT: catalase; QE: quercetin. a Kruskal–Wallis test was used for statistical analysis. Values are expressed as means + SD, n ¼ 10 for each group. b p < 0.01 compared with the control group. c p < 0.05 compared with control group.
Statistical analysis All statistical analyses were carried out using Statistical Package for Social Sciences software (SPSS for Windows, version 11.0; SPSS Inc., Chicago, Illinois, USA). All data were expressed as mean + SD. Differences in measured parameters among the three groups were analyzed with a nonparametric test (Kruskal–Wallis). Dual comparisons between groups exhibiting significant values were evaluated with Mann–Whitney U test. These differences were considered significant when p < 0.05.
Results Biochemical findings MDA levels of the tissues. MDA levels in the hippocampal tissue were found to be significantly higher in the Cd group than the control group. Treatment with QE prevented elevation of MDA levels significantly (Table 1). PC levels of the tissues. In the Cd group, there was a significant increase in protein oxidation levels in the hippocampal tissue, when compared with the control group. QE treatment significantly prevented protein oxidation (Table 1). SOD and CAT activities of the tissues. SOD and CAT activities in the hippocampal tissue was found to be significantly less in the Cd group than in the control group. The activities of both enzymes were kept at a level similar to the control group in Cd þ QE group (Table 1).
Histological findings In the control group, the morphology of neurons in CA2 region of the hippocampal tissue was normal
(Figure 1(a)). The most consistent findings in histologic sections of hippocampal neurons in Cd group stained with H&E were severe degenerative changes, shrunken cytoplasma, and extensively dark pyknotic nuclei (Figure 1(b)). Treatment with QE reduced the incidence of these degenerative changes in the hippocampal tissues and showed almost normal architecture similar to the control group (Figure 1(c)).
Immunohistochemical findings Light micrographs showed apoptotic hippocampal neurons after Cd exposure by caspase 3 immunohistochemistry (Figure 2). Mild caspase 3 immunoreactivity was observed in the cytoplasm of neurons in control rats CA2 region of the hippocampus (Figure 2(a)). The caspase 3 immunopositivity was increased in degenerating neurons in the CA2 region of the hippocampus following Cd exposure (Figure 2(b)). Treatment of QE markedly reduced the immunoreactivity of degenerating neurons after Cd exposure (Figure 2(c)). Moreover, the number of caspase 3 positive apoptotic neurons in the CA2 region of the hippocampus were increased significantly in Cd-treated rats compared with the control (p < 0.001) and Cd þ QE group (p < 0.01) rats’ hippocampal tissues (Table 2).
Discussion This experiment was carried out to evaluate the biochemical and histopathological changes in hippocampal tissues due to oxidative damage by Cd and protective effect of QE. Cd can disrupt the main structural components of the blood–brain and blood–cerebrospinal fluid barrier by injuring endothelial, epithelial, and glial cells and affecting the formation of tight junctions between barrier cells (Fleming et al., 2000; Virgintino et al.,
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Figure 1. Representative light microphotographs showing the histology of CA2 region of the hippocampus after Cd exposure. (a) In control group, normal hippocampal neurons are seen. (b) In Cd-treated rats, the severe degenerative changes in the cytoplasma and especially in the hippocampal cell nuclei of cells are seen. (c) Treatment with QE reduced the incidence of these degenerative changes and was less as compared to the Cd group (H&E, scale bar, 50 m). Cd: cadmium; QE: quercetin; H&E: hematoxylin and eosin.
2004). Then, Cd in circulation can enter the CNS and induce negative effects (Duval and Grubb, 1986). Cd has a long biological half-life (about 30 years in humans), which contributes to its accumulation in various tissues such as the brain (Usai et al., 1999). Cd is also toxic to neurons. The metal causes continuous elevation in intracellular calcium concentrations in neurons (Yoshida, 2001) and neuronal death by apoptosis or necrosis (Lopez et al., 2003). Cd has been reported to induce metallothionein expression in astrocytes (Rising et al., 1995). Cd can enter the body through inhalation, dermal exposure, and oral intake of contaminated food and drinking water. For example, electronic waste contains a number of toxic heavy metals, such as Cd, which can be released from ewastes through primitive recycling processes. Previously, Huo et al. (2007) reported that children living in e-waste dismantling site had significantly higher blood Cd levels as compared to those living in neighboring town. Brain aging is a risk factor for neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease. Oxidative damage plays a significant role in
brain aging (Johnson et al., 1999; Reiter, 1995), which induces overproduction of reactive oxygen species (ROS), decreases antioxidant enzyme activity and induces neuronal degeneration (Churchill et al., 2002; Colcombe et al., 2003). Oxidative stress represents an imbalance between the production of ROS including peroxides and free radicals, and the ability of biological system to readily detoxify the reactive intermediates or to repair the resulting damage (Sies, 1997). Disturbances in the normal redox state of tissues can cause toxic effects through the production of ROS that damage all components of the cell, including proteins, lipids, and DNA (Valko et al., 2007). Cells are protected against oxidative stress by an interacting network of antioxidant enzymes such as SOD and CAT (Sies, 1997). Many teratogenic mechanisms, including that of thalidomide have been linked to ROS production (Hansen et al., 2002). Cd has also been described as a potent inhibitor of antioxidant enzymes and closely associated with oxidative stress both in adult tissue (Shukla and Kumar, 2009) and in embryonic tissue (Paniagua-Castro et al., 2008). LPO is the process
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Figure 2. Representative light microphotographs showing apoptotic hippocampal neurons of CA2 region of the hippocampus after Cd exposure by caspase 3 immunohistochemistry. (a) In control rats, neuronal cells are moderately stained with the anticaspase 3 antibody. (b) In Cd-treated rats, the caspase 3 immunopositivity was strongly increased in CA2 neurons of the hippocampus after Cd exposure. (c) Treatment of QE markedly reduced the immunoreactivity of degenerating neurons after toluene exposure. (Immunoperoxidase, hematoxylin counterstain, scale bar, 50 m). Cd: cadmium; QE: quercetin. Table 2. The numbers (number per square micrometer) of apoptotic neurons (caspase 3 immunopositive) in the CA2 region of the hippocampal tissue of all groups.a Groups Control Cadmium Cadmium þ QE
Hippocampal tissue 6.2 + 0.8 31.1 + 4.6b 19.8 + 2.3c
QE: quercetin. a Kruskal-Wallis test was used for statistical analysis. Values are expressed as means + SD, n ¼ 10 for each group. b p < 0.001 compared with the control group. c p < 0.01 compared with control group.
of oxidative degradation of polyunsaturated fatty acids and its occurrence in biological membranes causes impaired membrane fluidity and inactivation of several membrane-bound enzymes (Goel et al., 2005). MDA is one of the major oxidation products of peroxidized polyunsaturated fatty acids, and thus increased MDA content is an important indicator of LPO (Demir et al., 2011). QE excerts its antioxidant action by scavenging free radicals, such as superoxide radicals generated by
xanthine/xanthine oxidase (Dok-Go et al., 2003). Additionally, QE exhibited peroxyl and hydroxyl freeradical scavenging activity (Kefalas et al., 2003). Free-radical scavenging effect of QE is due to its ability to chelate divalent cations. QE has the hepatoprotective (Renugadevi and Milton Prabu, 2009) and nephroprotective effect against the Cd-induced toxicity by increasing the methyl transferase and endothelial nitric oxide synthase expression (Morales et al., 2006). Additionally, a short time since it has been notified that QE can pass through the BBB of in situ models (2004). In addition, QE applies the protective effect in a stroke model induced by transient global ischemia (2006). QE significantly protected the neuronal cells from the oxidative stress-induced neurodegeneration in Alzheimer’s disease (Heo and Lee, 2004), decreased MDA level, improved activity of CAT and SOD (Mahesh and Menon, 2004). Furthermore, it was found that QE attenuated the neuronal death in the hippocampus resulting in improved learning and memory in arm maze test (Pu et al., 2007). Hence, these pieces of evidence point out the possibility that QE might exert an influence on the CNS.
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Therefore, we investigated the effects of QE on oxidative stress after Cd exposure. We show for the first time that Cd induced hippocampal tissue damage and apoptosis. In our current study, increases in MDA and PC levels, as well as decreases in SOD and CAT activity, tissue damage, and neuronal apoptosis induced by Cd detoxification were prevented by QE treatment, demonstrating that oxidative injury and apoptosis of the hippocampal tissue were attenuated by this treatment. To date, there is no information on the effect of QE on neuronal damage after Cd exposure in hippocampus. In a study, Mendez-Armenta et al. (2001) showed that there were degenerative changes that included pyknosis, necrosis, interstitial edema, and hyperchromatic cells in hippocampus of brain after Cd exposure. In another study, Gerspacher et al. (2009) reported that Cd exposure induces severe damage in primary cultures of both cortical neurons and glial cells, as demonstrated by vitality assays. Moreover, two different cell lines that we used as model of either neurons pheochromocytoma cells or brain capillary endothelial cells were also damaged by Cd. Our histological results show that Cd can cause diffuse neuronal damage. These results correlate well with the above literatures. The observed histological impairments by Cd have been recovered significantly by QE in QE þ Cd-treated rats indicating that QE is capable of preventing the neuronal damage induced by Cd. Therefore, it may be suggested that QE might inhibit Cd-induced hippocampal tissue damage. In other cellular systems, Cd produces either apoptotic or necrotic cell death depending on culture conditions as well as on the concentration and the duration of exposure. Apoptosis and necrosis are two morphologically and biochemically distinct processes of cell death, discernable based on cellular and organellar membrane integrity, nuclear fragmentation, enzymatic activation, and release of cellular content (Wyllie et al., 1980). Cd-induced apoptosis in vivo (Lag et al., 2002) and in vitro (Lasfer et al., 2008; Shih et al., 2003) was found to primarily involve the intrinsic mitochondrial-dependent pathway. Following chronic Cd exposure, there was an upregulation in gene expression of Bax (Fernandez et al., 2003; Shin et al., 2003), p53, p21, and a downregulation of Bcl-2 (Shin et al., 2003). Caspase 3, an effector caspase (Nicholson, 1999), has been shown to be critical in Cd-induced apoptosis in the above-mentioned studies. Kanter (2013) suggests that QE may be beneficial to neuronal apoptosis following Cd exposure in frontal cortex brain. In our current study, results supported
that the above-mentioned literatures reported on apoptosis in terms of neuronal survival. Moreover; the number of apoptotic neurons in hippocampal tissue of Cd-treated group was significantly more than both the control and Cd þ QE groups. We conclude that the natural flavonoid QE inhibits LPO, neuronal cell apoptosis, hippocampal neuronal cell damage, and raises antioxidant enzyme activity of the hippocampal tissue after Cd exposure. We believe that further preclinical research into the utility of QE may indicate its usefulness as a potential treatment on Cd-induced neurodegeneration. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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