Journal of Trace Elements in Medicine and Biology 31 (2015) 53–60
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TOXICOLOGY
Hesperidin ameliorates heavy metal induced toxicity mediated by oxidative stress in brain of Wistar rats Mohammad Haaris Ajmal Khan, Suhel Parvez ∗ Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
a r t i c l e
i n f o
Article history: Received 14 November 2014 Accepted 16 March 2015 Keywords: Cadmium Heavy metal Hesperidin Rats Neurotoxicity Antioxidant
a b s t r a c t Cadmium (Cd) induces neurotoxicity owing to its highly deleterious capacity to cross the blood brain barrier (BBB). Recent studies have provided insights on antioxidant properties of bioflavonoids which have emerged as potential therapeutic and nutraceutical agents. The aim of our study was to examine the hypothesis that hesperidin (HP) ameliorates oxidative stress and may have mitigatory effects in the extent of heavy metal-induced neurotoxicity. Cd (3 mg/kg body weight) was administered subcutaneously for 21 days while HP (40 mg/kg body weight) was administered orally once every day. The results of the current investigation demonstrate significant elevated levels of oxidative stress markers such as lipid peroxidation (LPO) and protein carbonyl (PC) along with significant depletion in the activity of non-enzymatic antioxidants like glutathione (GSH) and non-protein thiol (NP-SH) and enzymatic antioxidants in the Cd treated rats’ brain. Activity of neurotoxicity biomarkers such as acetylcholinesterase (AchE), monoamine oxidase (MAO) and total ATPase were also altered significantly and HP treatment significantly attenuated the altered levels of oxidative stress and neurotoxicity biomarkers while salvaging the antioxidant sentinels of cells to near normal levels thus exhibiting potent antioxidant and neuroprotective effects on the brain tissue against oxidative damage in Cd treated rodent model. © 2015 Elsevier GmbH. All rights reserved.
Introduction Heavy metals, a poorly defined group of elements which primarily includes transition metals, some metalloids, lanthanides and actinides, may enter human body and have previously been implicated in causing various adverse health effects. Research on heavy metals have established the prime routes of exposure to be ingestion of food plants cultivated on soils laden with these contaminants and water followed by inhalation of dust and air and topical exposure in agriculture, manufacturing, pharmaceutical and industrial settings [1,2]. The biological manifestations of heavy metal intoxication are linked to their chemical properties. One such adverse biological manifestation among many others is the neurotoxicity associated to the exposure of heavy metals which has lately caught the attention of researchers. Cadmium (Cd) is a serious environmental toxicant which is widely distributed and has the potential to affect cellular antioxidant defenses, damage oxidative DNA repair systems, play a vital role in differentiation and apoptosis with the underlying cause
∗ Corresponding author. Tel.: +91 11 26059688x5573; fax: +91 11 26059663. E-mail addresses: [email protected], [email protected] (S. Parvez). http://dx.doi.org/10.1016/j.jtemb.2015.03.002 0946-672X/© 2015 Elsevier GmbH. All rights reserved.
being the heightened production of reactive oxygen species (ROS) which may act as a signaling molecule in apoptosis [3–5]. Cd though widely recognized for its ROS generation potential is a non-redox active bivalent metal which produces free radicals indirectly by increasing the concentration of free Fe most likely by the latter’s replacement in various proteins as Cd itself has only one oxidation state and hence unable to generate free radical directly. Oxidative stress indirectly induced by cadmium manifests in the form of inhibition of antioxidative components of the cell such as superoxide dismutase (SOD) which may show both increase and decrease of levels, an inconsistency which is best explained by different exposure conditions [6,7]. This metal has been reported to alter the functioning of mitochondrial function too primarily through the mechanism of lipid peroxidation (LPO) of the mitochondrial membrane which results in the undermining of the mitochondrial membrane integrity [8]. ROS induced by Cd leads to mitochondrial membrane depolarization [9]. This loss of membrane potential is a crucial event in the intrinsic pathway of apoptosis as loss of potential causes mitochondrial pores and hence the opening of gates on cytochrome c which escapes to the cytoplasm initiating the programmed cell death pathway [10]. Several reports have been shown that Cd induces cell death through apoptosis in many tissues and cells [11–13].
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Among all others, brain is one of those organs which is highly susceptible to the adverse effects of oxidative stress as it bears plenty of polyunsaturated fatty acids in its membranous fence which are a soft target for LPO. Studies have shown that low levels of Cd cross through the brain barrier into the brain in adult rats and higher levels gain access if ethanol is used to deliver it to the brain and has the point of vantage to induce oxidative stress in brain cells. Thus Cd is able to induce neurotoxicity with a varied spectrum of alterations to the normal functioning of the neurons and other associated entities like action of neurotransmitters primarily depending on its highly deleterious capacity to cross the blood brain barrier (BBB). Extensive research is being done to evaluate several natural antioxidants for their therapeutic effects in heavy metals induced toxicities [14]. Flavonoids are natural substances that comprise a large group of naturally occurring compounds with low molecular weight and variable phenolic structures which are present in food and medicinal plants. One of the widely distributed and easily available flavonoid found abundantly in citrus fruits is hesperidin (HP). The antioxidant activity of HP has been, like all other flavonoids, attributed to its chemical structure as this flavonoid derives its antioxidant property from a hydroxyl group that it bears at the position 3 of the ring B. The pharmacological effects of HP have been repeatedly subjected to analysis in order to screen for its beneficial properties [15]. It has been found to possess anti-inflammation and analgesic activity along with the ability to reduce superoxide in in vitro electron transfer reaction. The pharmacological property of HP which bears special relevance to our study is that it has the capacity to cross the BBB just like other common flavonoids and produce effects on the CNS. Mammoth amounts of evidence have accumulated out of works of investigators which shows that flavonoids have effects on memory, cognition and neurodegeneration [16]. Studies show that flavonoids have potential to protect neurons against injury induced by neurotoxins and neuroinflammation and ability to improve cerebrovascular blood flow [17]. No study has still been reported where HP has been tested against Cd in rodent brain models. Hence the purpose of this study was to measure and analyze the ameliorative capacity of HP in case of cadmium induced neurotoxicity in adult male rats. Materials and methods Materials Bovine serum albumin (BSA), butylated hydroxy toluene (BHT), 1-chloro-2, 4-dinitrobenzene (CDNB), 5,5 -dithiobis (2nitrobenzoic acid) (DTNB), epinephrine, oxidized glutathione (GSSG), reduced glutathione (GSH), hydrogen peroxide (H2 O2 ), nicotinamide adenine dinucleotide phosphate (NADPH), ophosphoric acid (OPA), thiobarbituric acid (TBA), trichloro acetic acid (TCA) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). 2,4-Dinitrophenyl hydrazine (DNPH), ethylenediaminetetraacetic acid (EDTA), and sulfosalicylic acid (SSA) were purchased from Merck Limited (Mumbai, India). Guanidine hydrochloride and sodium azide were obtained from Hi-Media Labs (Mumbai, India). HP was obtained from Sigma Chemicals Co. and CdCl2 was purchased from Merck Limited. Animals In this study male Wistar rats (250–280 g body weight) were used. Rats were obtained from the animal house of Jamia Hamdard (Hamdard University). The standard guidelines of Institutional Animal Ethics Committee were obeyed during the whole experiment
and the study was approved by them. Rats were kept at temperature 30 ± 1 ◦ C with relative humidity at 65 ± 10% and at a photoperiod of 12 h light/dark cycle. Standard pellet rodent diet and water were provided to the animals ad libitum. Experimental design To evaluate the neurotoxicity caused by HP and the protective effect of CdCl2 under in vivo conditions, the animals were divided into four groups (n = 6 per group) Control, HP, CdCl2 and CdCl2 + HP groups. All groups were treated for a period of 21 days. HP was administered orally once every day at a dose of 40 mg/kg b.wt., while CdCl2 was injected subcutaneously once every day at a dose of 3 mg/kg b.wt. In CdCl2 + HP group, HP was administered prior to CdCl2 injection. Control group was treated both orally and subcutaneously with saline once every day. The following doses were selected on the basis of previous study and literature reports [18,19]. After the end of the experimental period all animals were sacrificed by decapitation. The whole brain were quickly excised, rinsed in ice-cold phosphate buffer and kept chilled until homogenization. Homogenate preparation The brain tissue were homogenized in 0.1 phosphate buffer, pH – 7.4 to obtain 10% homogenate using a Potter–Elevehjam homogenizer giving 6–8 strokes at medium speed keeping the sample under ice. Post mitochondrial supernatant (PMS) preparation Homogenate was subjected to differential centrifugation in refrigerated centrifuge at temperature of 4 ◦ C. It was centrifuged at 10,000 rpm for 20 min. The resulting pellet is the primary mitochondrial pellet and the supernatant is 10% post mitochondrial supernatant. PMS was used for the estimation of various biochemical analysis. Oxidative stress indices LPO LPO was measured using the procedure of Chaudhary and Parvez [20]. Determination of LPO is based on the thiobarbituric acid reacting species (TBARS), which largely include malondialdehyde. The rate of LPO was expressed as moles of TBARS formed/h/g tissue using a molar extinction coefficient of 1.56 × 105 M−1 cm−1 . PC The oxidative damage to proteins was measured according to the method described by Waseem and Parvez [21]. The quantification of carbonyl groups was based on the reaction with DNPH. DNPH reacts with protein carbonyls to produce the corresponding hydrazone. The carbonyl content was measured spectrophotometrically at 340 nm. The results were expressed as nmoles of DNPH incorporated/mg protein based on the molar extinction coefficient of 22,000 M−1 cm−1 . Non-enzymatic antioxidant assays GSH. GSH was assessed by the method of Ashafaq et al. [22]. The reaction is based on the fact that the thiol group of GSH reacts with the SH reagent (DTNB) to form thionitro benzoic acid. The reduced glutathione concentration was calculated as nmoles GSH/mg protein using a molar extinction coefficient of 1.36 × 104 M−1 cm−1. Non-protein thiol (NP-SH). NP-SH was assessed by the method of Ashafaq et al. [22] which measures the entire pool comprising
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majorly of GSH and other associated components. The reaction mixture consisted of 0.4 mL H2 O, 0.5 mL PMS (10%) and 0.1 mL of TCA (40%). It was centrifuged at 2000 rpm for 10–15 min. 0.5 mL of the supernatant was taken with 1 mL Tris buffer (0.4 M, pH 8.9) and 0.025 mL DTNB. The reaction mixture was read at 412 nm. 0.9 mL H2 O and 0.1 mL TCA were taken as blank. Enzymatic antioxidant assays GST. GST activity was measured by the method of Govil et al. [23]. The enzyme activity was calculated as nmoles of CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 × 103 M−1 cm−1 at 340 nm. GPx. GPx activity was assayed using the method of Chaudhary and Parvez [20]. The enzyme activity was calculated as nmoles of NADPH oxidized/min/mg protein using a molar extinction coefficient of 6.22 × 103 M−1 cm−1 at 340 nm. GR. GR activity was estimated by the method of Chaudhary and Parvez [20]. For GR activity measurement, NADPH consumption is monitored. The enzyme activity was calculated as NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22 M−1 cm−1 . GR activity was measured kinetically at 340 nm. Xanthine oxidase (XO). The activity of XO was assayed by the method of Chaudhary and Parvez [20]. The enzyme activity was calculated as nmoles of uric acid formed/min/mg protein, using a molar extinction coefficient of 12,200 M−1 cm−1 . AChE. AChE was estimated by using the method of Govil et al. [23]. The enzyme activity was calculated as nmoles of ATC hydrolyzed/min/mg protein using a molar extinction coefficient of 1.36 × 104 M−1 cm−1 . MAO. MAO was measured by using the method of Ashafaq et al. [22], based on oxidation of BAHC to benzaldehyde. The enzyme activity was calculated as moles of BAHC hydrolyzed/min/mg protein using molar extinction coefficient of 7.6925 M−1 cm−1 . Total ATPase. Total ATPase activity was measured as the release of inorganic phosphate (Pi ) by the method of Chaudhary and Parvez [20]. The activity was measured as g Pi liberated/min/mg protein. Protein determination Protein content in sample was estimated by the method of Lowry et al. [24] using BSA as standard. Histopathological studies Tissue of brain was fixed in 10% formalin solution. The fixed tissues were processed, embedded in paraffin and sectioned (5–6 m). The sections were stained with hematoxylin and eosin (H and E) and observed under microscope.
Fig. 1. Effect of CdCl2 (3 mg/kg b.wt.) and HP (40 mg/kg b.wt.) on (A) LPO level and (B) PC content in brain of rat. Each value represents mean ± SE (n = 6). LPO was measured as moles of TBARS formed/h/g tissue, whereas PC was measured as nmoles of DNPH incorporated/mg protein. Significant differences were indicated by (*p < 0.05), (**p < 0.01) and (***p < 0.001) when compared to the control group, and (## p < 0.01), (### p < 0.001) was used to show significant difference when compared to the CdCl2 exposed group.
Results Oxidative stress markers Effect on LPO Results shows significant increase in LPO (p < 0.01) in CdCl2 exposed group as compared to control group in the brain tissue of rats (Fig. 1A). Animals in group treated with HP along with simultaneous exposure to CdCl2 resulted in depletion of LPO level (p < 0.001) in comparison to CdCl2 alone group. The LPO level in HP alone group showed significant difference (p < 0.05) when compared with control animals.
Statistical analysis Results are expressed as mean ± standard error (SE). All data were analyzed using analysis of variance (ANOVA) followed by Tukey’s test. Values of p < 0.05 were considered as significant. All the statistical analyses were performed using graph pad prism 5 software (Graph Pad Software Inc., San Diego, CA, USA).
Effect on PC CdCl2 exposed group had significantly increased (p < 0.001) PC levels as compared with control (Fig. 1B). In CdCl2 + HP, the content of PC was significantly declined (p < 0.01) compared to that of the CdCl2 exposed group. There is no significant difference between HP treated and the control group.
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Enzymatic antioxidant capacity Effect on GST activity There is a significant decrease in GST activity (p < 0.001) in the CdCl2 exposed group with comparison to control group (Fig. 3A). In CdCl2 + HP group significant increase (p < 0.05) is seen as compared to CdCl2 group. The HP treatment group shows no significant difference in comparison to control group. Effect on GPx activity In the CdCl2 exposed group there is significant decrease (p < 0.001) in the activity of GPx in comparison to the control group (Fig. 3B). The CdCl2 + HP group shows significant (p < 0.01) increase in the GPx activity in comparison to the CdCl2 exposed group. There is no significant difference between the HP treated group in comparison to the control group. Effect on GR activity In comparison to the control group there is significant (p < 0.01) depletion in GR in the CdCl2 group, while there is significant (#p < 0.05) increase in GR activity in CdCl2 + HP group in comparison to the CdCl2 exposed group. The HP treated group shows no significant difference with comparison to control group. Effect on XO activity In the case of XO there is a significant (p < 0.001) elevation in the activity of XO in the CdCl2 exposed group in comparison to the control group (Fig. 3D). There is also significant (p < 0.01) decrease in the activity of XO in the CdCl2 + HP group in comparison to the CdCl2 exposed group. There is no significant difference in the HP treated group in comparison to the control group. Effect on neurotoxicity biomarkers
Fig. 2. Effect of CdCl2 (3 mg/kg b.wt.) and of HP (40 mg/kg b.wt.) on (A) GSH and (B) NP-SH level in brain of rat. Each value represents mean ± SE (n = 6). GSH was measured as nmol GSH/mg protein, and NP-SH as nmol NP-SH/mg protein. Significant differences were indicated by (**p < 0.01) when compared to the control group and (# p < 0.05), (## p < 0.01) was used to show significant increase when compared to the CdCl2 exposed group.
Non-enzymatic antioxidant activity Effect on GSH In CdCl2 exposed group, there is a significant decrease (p < 0.01) in the GSH level as compared to control group (Fig. 2A). Animals of the group treated with HP along with simultaneous exposure to CdCl2 , the GSH level was significantly increased (p < 0.01) as compared to the toxicant i.e. CdCl2 alone group. There is no significant difference between HP treated and control group.
Effect on NP-SH Biochemical analysis shows that in the CdCl2 exposed group there is significant (p < 0.01) decrease in the level of NP-SH in comparison to the control group (Fig. 2B). The CdCl2 + HP group shows significant (p < 0.05) increase in the NP-SH level in comparison to the CdCl2 exposed group. There is no significant difference between the HP treated group in comparison to the control group.
Effect on AChE activity Results thrown up by biochemical estimation show that in the CdCl2 exposed group there is significant (p < 0.01) decrease in the activity of AChE activity in comparison to the control group (Fig. 4A). The CdCl2 + HP group shows significant (p < 0.05) increase in the AChE activity in comparison to the CdCl2 exposed group. There is no significant difference between the HP treated group in comparison to the control group. Effect on MAO activity In the CdCl2 exposed group there is significant (p < 0.001) decrease in the activity of MAO activity in comparison to the control group (Fig. 4B). The CdCl2 + HP group shows significant (p < 0.01) increase in the MAO activity in comparison to the CdCl2 exposed group. There is no significant difference between the HP treated group in comparison to the control group. Effect on total ATPase activity Enzymatic estimation of ATPase depicts that in the CdCl2 exposed group there is significant (p < 0.01) decrease in the activity of total ATPase in comparison to the control group (Fig. 4C). The CdCl2 + HP group shows significant (p < 0.01) increase in the total ATPase activity in comparison to the CdCl2 exposed group. There is no significant difference between the HP treated group in comparison to the control group. Histological changes in brain Histopathologic changes in brain were investigated by hematoxylin–eosin staining. Cd intoxicated rats exhibited nuclear pyknosis, apoptosis and presence of vacuolated spaces as against
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Fig. 3. Effect of CdCl2 (3 mg/kg b.wt.) and HP (40 mg/kg b.wt.) on (A) GST (B) GR (c) GPx and (D) XO activity in brain of rat. Each value represents mean ± SE (n = 6). GST activity was measured as nmoles of CDNB conjugate formed/min/mg protein, GPx and GR activity were measured as nmoles of NADPH oxidized/min/mg protein and XO activity is measured in terms of nmol of uric acid formed/min/mg protein. Significant differences were indicated by (## p < 0.01) and (***p < 0.001) when compared to the control group and (# p < 0.05) and (## p < 0.01) was used to show significant increase when compared to the CdCl2 exposed group.
normal architecture shown by the brain of vehicle and melatonin control rats. Intact neurons were absent in that area. The corresponding area in the sections from the HP + CdCl2 group showed partial neuronal loss with presence of intact neurons in between the vacuolated spaces. Hesperidin treatment ameliorated neuronal abnormalities in the HP + CdCl2 group as compared with the CdCl2 group animals (Fig. 5). Discussion Oxidative stress, a detrimental cellular environment, comes into existence due to skewed equilibrium between pro-oxidants and antioxidants present inside the cell. This equilibrium could be disrupted by an unregulated and abnormal increase in the levels of any of the varied chemical entities broadly classified as free radicals or oxidants or conversely by an abnormal depreciation in the levels of the antioxidants, both enzymatic and non enzymatic. One of the direct implications of the elevated levels of oxidant is the consumption of the antioxidant entities in the attempt to neutralize the newly proliferated oxidants. Consequently, in the event of the amount of oxidants being beyond the scope of being reined in by the antioxidants, a stage arrives at which the adverse effects of heightened amounts of oxidants begin to manifest in the form other biochemical and cellular distortions. Hence we
can clearly comprehend that the constituents present on the two sides of the above mentioned equilibrium between oxidants and antioxidants are interlinked and form a complex network. Therapeutic interventions such as flavonoids which have the potential to influence these constituents across the spectrum of oxidants and antioxidants would have the ability to reverse the adverse effects to a large extent. This is precisely what we observe in this particular study where cadmium acts as an inducer of oxidative stress. Lipids along with proteins form the bulk of the biomembranes found in the cells. Lipids and proteins undergo oxidation in highly oxidized environment created as a result of elevated levels of free radicals and ROS. Oxidized forms of lipids called LPO act as markers of oxidative stress and a direct proportionality has been observed between the extent of oxidized environment inside the cell and level of LPO [25]. Our study shows elevated levels of LPO under the exposure of Cd probably by stimulating the production of superoxide anions. Cd has been previously implicated in peroxidation of lipids and this has been referred to be the primary mechanism involved in the manifestation of its toxicity [26]. Our study throws up results where we see reduced levels of LPO by HP by countering the effects of Cd induced oxidative stress which is on the expected lines with the results of other investigators. Elevated levels of LPO also impact the performance of other non-enzymatic antioxidants
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Fig. 4. Effect of CdCl2 (3 mg/kg b.wt.) and HP (40 mg/kg b.wt.) on (A) AChE (B) MAO and (C) total ATPase activity in brain of rats. Each value represents mean ± SE (n = 6). AChE activity is measured in terms of nmol ATC hydrolyzed/min/mg protein, MAO activity is measured in terms of mol BAHC hydrolyzed/min/mg protein and total ATPase activity is measured in terms of g Pi liberated/min/mg protein. Significant difference in comparison to the control group is indicated by (**p < 0.01) and (***p < 0.001) and significant difference in comparison to the CdCl2 exposed group is indicated by (# p < 0.05) and (## p < 0.01).
like GSH which get consumed in the effort to reverse the oxidized state of lipids. The fact that oxidation of cellular components is one of the prime mechanisms behind Cd toxicity is complemented by the elevated levels of oxidized proteins or protein carbonyls as demonstrated by our study which also derives support from other similar studies [27]. HP with its known antioxidant properties [28] ameliorates the effect of ROS and free radicals, produced by Cd, on proteins. The results of our study are consistent with these observations and HP reduces protein carbonyl levels against the action of Cd in brain of animals. One of the properties of Cd which synergizes its toxic potential is its affinity for thiols which makes the major thiol antioxidant glutathione (GSH) as its primary target. Cd can form covalent bonds with various cellular thiols such as GSH and metallothioneins (MT) [29]. In case Cd is administered externally it is taken up by the liver and bound to GSH and MT. It has long been acknowledged that sulfhydryl-containing compounds have the ability to chelate metals. The binding of Cd with sulfhydryl groups of cysteine moiety of GSH forms a thermodynamically stable and inert mercapeptide complex. These modifications of amino acids which alter the structure of the antioxidant and render it inactive leads to the overwhelming of GSH levels and hence compromise the antioxidant defense system in case of high amount of cadmium exposure [30]. This depletion in the level of free GSH leads to elevation in the production of ROS such as superoxide, hydroxyl radicals and hydrogen peroxide causing damage consistent with oxidative stress [31]. Present study replicates these results in the sense that cadmium exposure led to the inhibition of GSH levels in the brain tissue along with other GSH metabolizing enzymes like GST and GR. GR is the enzyme responsible for the reversal of oxidized GSH or GSSG back to the reduced state while GST is involved with the action of neutralizing oxidative agents like the superoxide radicals by conjugating these electrophiles with GSH. Depletion in the levels of GSH is expected to influence the activity of these GSH metabolizing enzymes as they get overwhelmed and inhibited by cadmium induced oxidative stress in absence of sufficient GSH ([39]). Depleted levels of GSH also makes the lipids of the membrane more susceptible to oxidation and hence in a way initiates a vicious cycle. The reduced levels of GSH and inhibited activity of GST and GR under the exposure of Cd is reversed by the administration of natural flavonoids like HP which agrees with the results of earlier reported experiments [32]. The mechanism of HP action in ameliorating GSH depletion is attributed to the metal chelating, free radical scavenging and antioxidant property of this flavonoid by which it prevents the binding or conjugation of Cd to the thiol groups of the GSH and thereby minimizes the consumption of these antioxidants, thus restoring their levels [33]. GPx is responsible for countering low levels of oxidative stress and has been reported to show an increase in activity in case of acute (24 h) intraperitoneal exposure to cadmium while decreased GPx levels were reported after chronic cadmium exposure in liver and kidney of mice and rats [34,35]. The alteration and toxicity of Cd was also demonstrated in the neurotoxicity specific biomarkers like ATPase, AChE and MAO in our study. The ATPase which includes enzymes like Na+ /K+ ATPase, responsible for neural excitability and energy production and Mg2+ ATPase which has the job of maintaining the intracellular levels of Mg2+ in the brain are observed to be inhibited under the exposure of Cd induced toxic insults. The inhibition of ATPase by Cd in the brain has been reported earlier [36] and has been explained to be occurring due to the disruption of membrane integrity and loss of membrane potential due to the peroxidation of the membrane lipids ([40]). It could also be from the formation of Cd-ATPase complexes through SH group of enzyme and increased oxidative stress [19]. HP has the potential to prevent the inhibition of ATPase by Cd as is seen in the results of
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Fig. 5. Histopathological analysis of control, CdCl2 , HP + CdCl2 , and HP groups. Panel A shows brain histopathology of control animals with uniform distribution of neurons. Panel B exhibits brain sections of CdCl2 exposed group showing degenerated neurons along with vacuolated spaces. Panel C shows histology of brain sections of HP pretreated CdCl2 exposed animals where there is only partial neuronal loss with more intact neurons. Panel D depicts HP treated rats resembling normal histology of brain (magnification for all images, 100×).
our study possibly by preventing the peroxidation of the membrane lipids which has also been validated by previous reports [19,37]. Many neurons in the human body secrete acetylcholine at the synapses which acts as neurotransmitters by diffusing across the post-synaptic membranes to transmit the impulse or information. The termination of acetylcholine is carried out by an enzyme called acetylcholinesterase which breaks it down to choline and acetic acid by adding water to the parent biomolecule. Inhibition of this enzyme causes accumulation of acetylcholine which might occur because of free radical production [38] leading to the conditions of cholinergic hyperactivity, convulsion and status epilepticus. The decreased level of AChE in plasma and brain might be one of the indicators for Cd induced hurdle in brain. Natural antioxidants have been found to ameliorate the inhibition of AChE in the case Cd induced neurotoxicity [14,27] and on similar lines HP has been observed to reduce the inhibition of AChE in the brain of rats exposed to Cd. These observations confirm the beliefs that Cd causes toxicity in brain through certain mechanisms which target those biochemical systems which are specifically found in the neurons and cells which inhabit the brain and HP as a high value potential therapeutic agent is up to the task of ameliorating these specialized toxic effects of Cd in the brain. Histological examination of the brain tissue reveals that Cd intoxication caused abnormal structural changes in the brain tissue including apoptosis and vacuolization. With reference to histopathological observation, observable difference has been observed between Cd and HP pre-treated Cd intoxicated rats as there is only partial degeneration of neurons which indicates that HP is capable of preventing the neuronal damage induced by Cd. Therefore, it may be suggested that HP might inhibit Cd induced brain damage.
Conclusion Taking an overview of the results obtained from the study while simultaneously keeping in perspective the background data we can conclude that Cd has various mechanisms to effect toxicity like by peroxidation of lipids, conjugation of thiol groups which would render GSH like antioxidants ineffective and loss of membrane potential and since it has the potential to cross the BBB it is well placed to cause neurotoxicity elucidated by the alteration in neurotoxic markers in the present study but HP has the potential to mitigate the toxic insults of Cd in the brain by varied mechanism involving chelation of Cd, prevention of loss of membrane integrity, protection of thiol containing groups but the underlying property which supplements all these protective actions is its potential as an antioxidant and just like its functional opponent, Cd, it too can cross the BBB and thus HP appears to possess the prospective therapeutic potential which could be harnessed to counter the neurotoxic manifestations of Cd toxicity in the event of an exposure.
Conflict of interests The authors declare that there is no conflict of interests.
Acknowledgment The Grant (no. F. 30-1/2013(SA-II)/RA-2012-14-GE-WES2400), received as Research Award (2012-14) from the University Grants Commission, New Delhi, Government of India to Dr. Suhel Parvez, is thankfully acknowledged.
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Contents lists available at ScienceDirect
Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.com/locate/jtemb
TOXICOLOGY
Hesperidin ameliorates heavy metal induced toxicity mediated by oxidative stress in brain of Wistar rats Mohammad Haaris Ajmal Khan, Suhel Parvez ∗ Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
a r t i c l e
i n f o
Article history: Received 14 November 2014 Accepted 16 March 2015 Keywords: Cadmium Heavy metal Hesperidin Rats Neurotoxicity Antioxidant
a b s t r a c t Cadmium (Cd) induces neurotoxicity owing to its highly deleterious capacity to cross the blood brain barrier (BBB). Recent studies have provided insights on antioxidant properties of bioflavonoids which have emerged as potential therapeutic and nutraceutical agents. The aim of our study was to examine the hypothesis that hesperidin (HP) ameliorates oxidative stress and may have mitigatory effects in the extent of heavy metal-induced neurotoxicity. Cd (3 mg/kg body weight) was administered subcutaneously for 21 days while HP (40 mg/kg body weight) was administered orally once every day. The results of the current investigation demonstrate significant elevated levels of oxidative stress markers such as lipid peroxidation (LPO) and protein carbonyl (PC) along with significant depletion in the activity of non-enzymatic antioxidants like glutathione (GSH) and non-protein thiol (NP-SH) and enzymatic antioxidants in the Cd treated rats’ brain. Activity of neurotoxicity biomarkers such as acetylcholinesterase (AchE), monoamine oxidase (MAO) and total ATPase were also altered significantly and HP treatment significantly attenuated the altered levels of oxidative stress and neurotoxicity biomarkers while salvaging the antioxidant sentinels of cells to near normal levels thus exhibiting potent antioxidant and neuroprotective effects on the brain tissue against oxidative damage in Cd treated rodent model. © 2015 Elsevier GmbH. All rights reserved.
Introduction Heavy metals, a poorly defined group of elements which primarily includes transition metals, some metalloids, lanthanides and actinides, may enter human body and have previously been implicated in causing various adverse health effects. Research on heavy metals have established the prime routes of exposure to be ingestion of food plants cultivated on soils laden with these contaminants and water followed by inhalation of dust and air and topical exposure in agriculture, manufacturing, pharmaceutical and industrial settings [1,2]. The biological manifestations of heavy metal intoxication are linked to their chemical properties. One such adverse biological manifestation among many others is the neurotoxicity associated to the exposure of heavy metals which has lately caught the attention of researchers. Cadmium (Cd) is a serious environmental toxicant which is widely distributed and has the potential to affect cellular antioxidant defenses, damage oxidative DNA repair systems, play a vital role in differentiation and apoptosis with the underlying cause
∗ Corresponding author. Tel.: +91 11 26059688x5573; fax: +91 11 26059663. E-mail addresses: [email protected], [email protected] (S. Parvez). http://dx.doi.org/10.1016/j.jtemb.2015.03.002 0946-672X/© 2015 Elsevier GmbH. All rights reserved.
being the heightened production of reactive oxygen species (ROS) which may act as a signaling molecule in apoptosis [3–5]. Cd though widely recognized for its ROS generation potential is a non-redox active bivalent metal which produces free radicals indirectly by increasing the concentration of free Fe most likely by the latter’s replacement in various proteins as Cd itself has only one oxidation state and hence unable to generate free radical directly. Oxidative stress indirectly induced by cadmium manifests in the form of inhibition of antioxidative components of the cell such as superoxide dismutase (SOD) which may show both increase and decrease of levels, an inconsistency which is best explained by different exposure conditions [6,7]. This metal has been reported to alter the functioning of mitochondrial function too primarily through the mechanism of lipid peroxidation (LPO) of the mitochondrial membrane which results in the undermining of the mitochondrial membrane integrity [8]. ROS induced by Cd leads to mitochondrial membrane depolarization [9]. This loss of membrane potential is a crucial event in the intrinsic pathway of apoptosis as loss of potential causes mitochondrial pores and hence the opening of gates on cytochrome c which escapes to the cytoplasm initiating the programmed cell death pathway [10]. Several reports have been shown that Cd induces cell death through apoptosis in many tissues and cells [11–13].
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Among all others, brain is one of those organs which is highly susceptible to the adverse effects of oxidative stress as it bears plenty of polyunsaturated fatty acids in its membranous fence which are a soft target for LPO. Studies have shown that low levels of Cd cross through the brain barrier into the brain in adult rats and higher levels gain access if ethanol is used to deliver it to the brain and has the point of vantage to induce oxidative stress in brain cells. Thus Cd is able to induce neurotoxicity with a varied spectrum of alterations to the normal functioning of the neurons and other associated entities like action of neurotransmitters primarily depending on its highly deleterious capacity to cross the blood brain barrier (BBB). Extensive research is being done to evaluate several natural antioxidants for their therapeutic effects in heavy metals induced toxicities [14]. Flavonoids are natural substances that comprise a large group of naturally occurring compounds with low molecular weight and variable phenolic structures which are present in food and medicinal plants. One of the widely distributed and easily available flavonoid found abundantly in citrus fruits is hesperidin (HP). The antioxidant activity of HP has been, like all other flavonoids, attributed to its chemical structure as this flavonoid derives its antioxidant property from a hydroxyl group that it bears at the position 3 of the ring B. The pharmacological effects of HP have been repeatedly subjected to analysis in order to screen for its beneficial properties [15]. It has been found to possess anti-inflammation and analgesic activity along with the ability to reduce superoxide in in vitro electron transfer reaction. The pharmacological property of HP which bears special relevance to our study is that it has the capacity to cross the BBB just like other common flavonoids and produce effects on the CNS. Mammoth amounts of evidence have accumulated out of works of investigators which shows that flavonoids have effects on memory, cognition and neurodegeneration [16]. Studies show that flavonoids have potential to protect neurons against injury induced by neurotoxins and neuroinflammation and ability to improve cerebrovascular blood flow [17]. No study has still been reported where HP has been tested against Cd in rodent brain models. Hence the purpose of this study was to measure and analyze the ameliorative capacity of HP in case of cadmium induced neurotoxicity in adult male rats. Materials and methods Materials Bovine serum albumin (BSA), butylated hydroxy toluene (BHT), 1-chloro-2, 4-dinitrobenzene (CDNB), 5,5 -dithiobis (2nitrobenzoic acid) (DTNB), epinephrine, oxidized glutathione (GSSG), reduced glutathione (GSH), hydrogen peroxide (H2 O2 ), nicotinamide adenine dinucleotide phosphate (NADPH), ophosphoric acid (OPA), thiobarbituric acid (TBA), trichloro acetic acid (TCA) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). 2,4-Dinitrophenyl hydrazine (DNPH), ethylenediaminetetraacetic acid (EDTA), and sulfosalicylic acid (SSA) were purchased from Merck Limited (Mumbai, India). Guanidine hydrochloride and sodium azide were obtained from Hi-Media Labs (Mumbai, India). HP was obtained from Sigma Chemicals Co. and CdCl2 was purchased from Merck Limited. Animals In this study male Wistar rats (250–280 g body weight) were used. Rats were obtained from the animal house of Jamia Hamdard (Hamdard University). The standard guidelines of Institutional Animal Ethics Committee were obeyed during the whole experiment
and the study was approved by them. Rats were kept at temperature 30 ± 1 ◦ C with relative humidity at 65 ± 10% and at a photoperiod of 12 h light/dark cycle. Standard pellet rodent diet and water were provided to the animals ad libitum. Experimental design To evaluate the neurotoxicity caused by HP and the protective effect of CdCl2 under in vivo conditions, the animals were divided into four groups (n = 6 per group) Control, HP, CdCl2 and CdCl2 + HP groups. All groups were treated for a period of 21 days. HP was administered orally once every day at a dose of 40 mg/kg b.wt., while CdCl2 was injected subcutaneously once every day at a dose of 3 mg/kg b.wt. In CdCl2 + HP group, HP was administered prior to CdCl2 injection. Control group was treated both orally and subcutaneously with saline once every day. The following doses were selected on the basis of previous study and literature reports [18,19]. After the end of the experimental period all animals were sacrificed by decapitation. The whole brain were quickly excised, rinsed in ice-cold phosphate buffer and kept chilled until homogenization. Homogenate preparation The brain tissue were homogenized in 0.1 phosphate buffer, pH – 7.4 to obtain 10% homogenate using a Potter–Elevehjam homogenizer giving 6–8 strokes at medium speed keeping the sample under ice. Post mitochondrial supernatant (PMS) preparation Homogenate was subjected to differential centrifugation in refrigerated centrifuge at temperature of 4 ◦ C. It was centrifuged at 10,000 rpm for 20 min. The resulting pellet is the primary mitochondrial pellet and the supernatant is 10% post mitochondrial supernatant. PMS was used for the estimation of various biochemical analysis. Oxidative stress indices LPO LPO was measured using the procedure of Chaudhary and Parvez [20]. Determination of LPO is based on the thiobarbituric acid reacting species (TBARS), which largely include malondialdehyde. The rate of LPO was expressed as moles of TBARS formed/h/g tissue using a molar extinction coefficient of 1.56 × 105 M−1 cm−1 . PC The oxidative damage to proteins was measured according to the method described by Waseem and Parvez [21]. The quantification of carbonyl groups was based on the reaction with DNPH. DNPH reacts with protein carbonyls to produce the corresponding hydrazone. The carbonyl content was measured spectrophotometrically at 340 nm. The results were expressed as nmoles of DNPH incorporated/mg protein based on the molar extinction coefficient of 22,000 M−1 cm−1 . Non-enzymatic antioxidant assays GSH. GSH was assessed by the method of Ashafaq et al. [22]. The reaction is based on the fact that the thiol group of GSH reacts with the SH reagent (DTNB) to form thionitro benzoic acid. The reduced glutathione concentration was calculated as nmoles GSH/mg protein using a molar extinction coefficient of 1.36 × 104 M−1 cm−1. Non-protein thiol (NP-SH). NP-SH was assessed by the method of Ashafaq et al. [22] which measures the entire pool comprising
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majorly of GSH and other associated components. The reaction mixture consisted of 0.4 mL H2 O, 0.5 mL PMS (10%) and 0.1 mL of TCA (40%). It was centrifuged at 2000 rpm for 10–15 min. 0.5 mL of the supernatant was taken with 1 mL Tris buffer (0.4 M, pH 8.9) and 0.025 mL DTNB. The reaction mixture was read at 412 nm. 0.9 mL H2 O and 0.1 mL TCA were taken as blank. Enzymatic antioxidant assays GST. GST activity was measured by the method of Govil et al. [23]. The enzyme activity was calculated as nmoles of CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 × 103 M−1 cm−1 at 340 nm. GPx. GPx activity was assayed using the method of Chaudhary and Parvez [20]. The enzyme activity was calculated as nmoles of NADPH oxidized/min/mg protein using a molar extinction coefficient of 6.22 × 103 M−1 cm−1 at 340 nm. GR. GR activity was estimated by the method of Chaudhary and Parvez [20]. For GR activity measurement, NADPH consumption is monitored. The enzyme activity was calculated as NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22 M−1 cm−1 . GR activity was measured kinetically at 340 nm. Xanthine oxidase (XO). The activity of XO was assayed by the method of Chaudhary and Parvez [20]. The enzyme activity was calculated as nmoles of uric acid formed/min/mg protein, using a molar extinction coefficient of 12,200 M−1 cm−1 . AChE. AChE was estimated by using the method of Govil et al. [23]. The enzyme activity was calculated as nmoles of ATC hydrolyzed/min/mg protein using a molar extinction coefficient of 1.36 × 104 M−1 cm−1 . MAO. MAO was measured by using the method of Ashafaq et al. [22], based on oxidation of BAHC to benzaldehyde. The enzyme activity was calculated as moles of BAHC hydrolyzed/min/mg protein using molar extinction coefficient of 7.6925 M−1 cm−1 . Total ATPase. Total ATPase activity was measured as the release of inorganic phosphate (Pi ) by the method of Chaudhary and Parvez [20]. The activity was measured as g Pi liberated/min/mg protein. Protein determination Protein content in sample was estimated by the method of Lowry et al. [24] using BSA as standard. Histopathological studies Tissue of brain was fixed in 10% formalin solution. The fixed tissues were processed, embedded in paraffin and sectioned (5–6 m). The sections were stained with hematoxylin and eosin (H and E) and observed under microscope.
Fig. 1. Effect of CdCl2 (3 mg/kg b.wt.) and HP (40 mg/kg b.wt.) on (A) LPO level and (B) PC content in brain of rat. Each value represents mean ± SE (n = 6). LPO was measured as moles of TBARS formed/h/g tissue, whereas PC was measured as nmoles of DNPH incorporated/mg protein. Significant differences were indicated by (*p < 0.05), (**p < 0.01) and (***p < 0.001) when compared to the control group, and (## p < 0.01), (### p < 0.001) was used to show significant difference when compared to the CdCl2 exposed group.
Results Oxidative stress markers Effect on LPO Results shows significant increase in LPO (p < 0.01) in CdCl2 exposed group as compared to control group in the brain tissue of rats (Fig. 1A). Animals in group treated with HP along with simultaneous exposure to CdCl2 resulted in depletion of LPO level (p < 0.001) in comparison to CdCl2 alone group. The LPO level in HP alone group showed significant difference (p < 0.05) when compared with control animals.
Statistical analysis Results are expressed as mean ± standard error (SE). All data were analyzed using analysis of variance (ANOVA) followed by Tukey’s test. Values of p < 0.05 were considered as significant. All the statistical analyses were performed using graph pad prism 5 software (Graph Pad Software Inc., San Diego, CA, USA).
Effect on PC CdCl2 exposed group had significantly increased (p < 0.001) PC levels as compared with control (Fig. 1B). In CdCl2 + HP, the content of PC was significantly declined (p < 0.01) compared to that of the CdCl2 exposed group. There is no significant difference between HP treated and the control group.
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Enzymatic antioxidant capacity Effect on GST activity There is a significant decrease in GST activity (p < 0.001) in the CdCl2 exposed group with comparison to control group (Fig. 3A). In CdCl2 + HP group significant increase (p < 0.05) is seen as compared to CdCl2 group. The HP treatment group shows no significant difference in comparison to control group. Effect on GPx activity In the CdCl2 exposed group there is significant decrease (p < 0.001) in the activity of GPx in comparison to the control group (Fig. 3B). The CdCl2 + HP group shows significant (p < 0.01) increase in the GPx activity in comparison to the CdCl2 exposed group. There is no significant difference between the HP treated group in comparison to the control group. Effect on GR activity In comparison to the control group there is significant (p < 0.01) depletion in GR in the CdCl2 group, while there is significant (#p < 0.05) increase in GR activity in CdCl2 + HP group in comparison to the CdCl2 exposed group. The HP treated group shows no significant difference with comparison to control group. Effect on XO activity In the case of XO there is a significant (p < 0.001) elevation in the activity of XO in the CdCl2 exposed group in comparison to the control group (Fig. 3D). There is also significant (p < 0.01) decrease in the activity of XO in the CdCl2 + HP group in comparison to the CdCl2 exposed group. There is no significant difference in the HP treated group in comparison to the control group. Effect on neurotoxicity biomarkers
Fig. 2. Effect of CdCl2 (3 mg/kg b.wt.) and of HP (40 mg/kg b.wt.) on (A) GSH and (B) NP-SH level in brain of rat. Each value represents mean ± SE (n = 6). GSH was measured as nmol GSH/mg protein, and NP-SH as nmol NP-SH/mg protein. Significant differences were indicated by (**p < 0.01) when compared to the control group and (# p < 0.05), (## p < 0.01) was used to show significant increase when compared to the CdCl2 exposed group.
Non-enzymatic antioxidant activity Effect on GSH In CdCl2 exposed group, there is a significant decrease (p < 0.01) in the GSH level as compared to control group (Fig. 2A). Animals of the group treated with HP along with simultaneous exposure to CdCl2 , the GSH level was significantly increased (p < 0.01) as compared to the toxicant i.e. CdCl2 alone group. There is no significant difference between HP treated and control group.
Effect on NP-SH Biochemical analysis shows that in the CdCl2 exposed group there is significant (p < 0.01) decrease in the level of NP-SH in comparison to the control group (Fig. 2B). The CdCl2 + HP group shows significant (p < 0.05) increase in the NP-SH level in comparison to the CdCl2 exposed group. There is no significant difference between the HP treated group in comparison to the control group.
Effect on AChE activity Results thrown up by biochemical estimation show that in the CdCl2 exposed group there is significant (p < 0.01) decrease in the activity of AChE activity in comparison to the control group (Fig. 4A). The CdCl2 + HP group shows significant (p < 0.05) increase in the AChE activity in comparison to the CdCl2 exposed group. There is no significant difference between the HP treated group in comparison to the control group. Effect on MAO activity In the CdCl2 exposed group there is significant (p < 0.001) decrease in the activity of MAO activity in comparison to the control group (Fig. 4B). The CdCl2 + HP group shows significant (p < 0.01) increase in the MAO activity in comparison to the CdCl2 exposed group. There is no significant difference between the HP treated group in comparison to the control group. Effect on total ATPase activity Enzymatic estimation of ATPase depicts that in the CdCl2 exposed group there is significant (p < 0.01) decrease in the activity of total ATPase in comparison to the control group (Fig. 4C). The CdCl2 + HP group shows significant (p < 0.01) increase in the total ATPase activity in comparison to the CdCl2 exposed group. There is no significant difference between the HP treated group in comparison to the control group. Histological changes in brain Histopathologic changes in brain were investigated by hematoxylin–eosin staining. Cd intoxicated rats exhibited nuclear pyknosis, apoptosis and presence of vacuolated spaces as against
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Fig. 3. Effect of CdCl2 (3 mg/kg b.wt.) and HP (40 mg/kg b.wt.) on (A) GST (B) GR (c) GPx and (D) XO activity in brain of rat. Each value represents mean ± SE (n = 6). GST activity was measured as nmoles of CDNB conjugate formed/min/mg protein, GPx and GR activity were measured as nmoles of NADPH oxidized/min/mg protein and XO activity is measured in terms of nmol of uric acid formed/min/mg protein. Significant differences were indicated by (## p < 0.01) and (***p < 0.001) when compared to the control group and (# p < 0.05) and (## p < 0.01) was used to show significant increase when compared to the CdCl2 exposed group.
normal architecture shown by the brain of vehicle and melatonin control rats. Intact neurons were absent in that area. The corresponding area in the sections from the HP + CdCl2 group showed partial neuronal loss with presence of intact neurons in between the vacuolated spaces. Hesperidin treatment ameliorated neuronal abnormalities in the HP + CdCl2 group as compared with the CdCl2 group animals (Fig. 5). Discussion Oxidative stress, a detrimental cellular environment, comes into existence due to skewed equilibrium between pro-oxidants and antioxidants present inside the cell. This equilibrium could be disrupted by an unregulated and abnormal increase in the levels of any of the varied chemical entities broadly classified as free radicals or oxidants or conversely by an abnormal depreciation in the levels of the antioxidants, both enzymatic and non enzymatic. One of the direct implications of the elevated levels of oxidant is the consumption of the antioxidant entities in the attempt to neutralize the newly proliferated oxidants. Consequently, in the event of the amount of oxidants being beyond the scope of being reined in by the antioxidants, a stage arrives at which the adverse effects of heightened amounts of oxidants begin to manifest in the form other biochemical and cellular distortions. Hence we
can clearly comprehend that the constituents present on the two sides of the above mentioned equilibrium between oxidants and antioxidants are interlinked and form a complex network. Therapeutic interventions such as flavonoids which have the potential to influence these constituents across the spectrum of oxidants and antioxidants would have the ability to reverse the adverse effects to a large extent. This is precisely what we observe in this particular study where cadmium acts as an inducer of oxidative stress. Lipids along with proteins form the bulk of the biomembranes found in the cells. Lipids and proteins undergo oxidation in highly oxidized environment created as a result of elevated levels of free radicals and ROS. Oxidized forms of lipids called LPO act as markers of oxidative stress and a direct proportionality has been observed between the extent of oxidized environment inside the cell and level of LPO [25]. Our study shows elevated levels of LPO under the exposure of Cd probably by stimulating the production of superoxide anions. Cd has been previously implicated in peroxidation of lipids and this has been referred to be the primary mechanism involved in the manifestation of its toxicity [26]. Our study throws up results where we see reduced levels of LPO by HP by countering the effects of Cd induced oxidative stress which is on the expected lines with the results of other investigators. Elevated levels of LPO also impact the performance of other non-enzymatic antioxidants
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Fig. 4. Effect of CdCl2 (3 mg/kg b.wt.) and HP (40 mg/kg b.wt.) on (A) AChE (B) MAO and (C) total ATPase activity in brain of rats. Each value represents mean ± SE (n = 6). AChE activity is measured in terms of nmol ATC hydrolyzed/min/mg protein, MAO activity is measured in terms of mol BAHC hydrolyzed/min/mg protein and total ATPase activity is measured in terms of g Pi liberated/min/mg protein. Significant difference in comparison to the control group is indicated by (**p < 0.01) and (***p < 0.001) and significant difference in comparison to the CdCl2 exposed group is indicated by (# p < 0.05) and (## p < 0.01).
like GSH which get consumed in the effort to reverse the oxidized state of lipids. The fact that oxidation of cellular components is one of the prime mechanisms behind Cd toxicity is complemented by the elevated levels of oxidized proteins or protein carbonyls as demonstrated by our study which also derives support from other similar studies [27]. HP with its known antioxidant properties [28] ameliorates the effect of ROS and free radicals, produced by Cd, on proteins. The results of our study are consistent with these observations and HP reduces protein carbonyl levels against the action of Cd in brain of animals. One of the properties of Cd which synergizes its toxic potential is its affinity for thiols which makes the major thiol antioxidant glutathione (GSH) as its primary target. Cd can form covalent bonds with various cellular thiols such as GSH and metallothioneins (MT) [29]. In case Cd is administered externally it is taken up by the liver and bound to GSH and MT. It has long been acknowledged that sulfhydryl-containing compounds have the ability to chelate metals. The binding of Cd with sulfhydryl groups of cysteine moiety of GSH forms a thermodynamically stable and inert mercapeptide complex. These modifications of amino acids which alter the structure of the antioxidant and render it inactive leads to the overwhelming of GSH levels and hence compromise the antioxidant defense system in case of high amount of cadmium exposure [30]. This depletion in the level of free GSH leads to elevation in the production of ROS such as superoxide, hydroxyl radicals and hydrogen peroxide causing damage consistent with oxidative stress [31]. Present study replicates these results in the sense that cadmium exposure led to the inhibition of GSH levels in the brain tissue along with other GSH metabolizing enzymes like GST and GR. GR is the enzyme responsible for the reversal of oxidized GSH or GSSG back to the reduced state while GST is involved with the action of neutralizing oxidative agents like the superoxide radicals by conjugating these electrophiles with GSH. Depletion in the levels of GSH is expected to influence the activity of these GSH metabolizing enzymes as they get overwhelmed and inhibited by cadmium induced oxidative stress in absence of sufficient GSH ([39]). Depleted levels of GSH also makes the lipids of the membrane more susceptible to oxidation and hence in a way initiates a vicious cycle. The reduced levels of GSH and inhibited activity of GST and GR under the exposure of Cd is reversed by the administration of natural flavonoids like HP which agrees with the results of earlier reported experiments [32]. The mechanism of HP action in ameliorating GSH depletion is attributed to the metal chelating, free radical scavenging and antioxidant property of this flavonoid by which it prevents the binding or conjugation of Cd to the thiol groups of the GSH and thereby minimizes the consumption of these antioxidants, thus restoring their levels [33]. GPx is responsible for countering low levels of oxidative stress and has been reported to show an increase in activity in case of acute (24 h) intraperitoneal exposure to cadmium while decreased GPx levels were reported after chronic cadmium exposure in liver and kidney of mice and rats [34,35]. The alteration and toxicity of Cd was also demonstrated in the neurotoxicity specific biomarkers like ATPase, AChE and MAO in our study. The ATPase which includes enzymes like Na+ /K+ ATPase, responsible for neural excitability and energy production and Mg2+ ATPase which has the job of maintaining the intracellular levels of Mg2+ in the brain are observed to be inhibited under the exposure of Cd induced toxic insults. The inhibition of ATPase by Cd in the brain has been reported earlier [36] and has been explained to be occurring due to the disruption of membrane integrity and loss of membrane potential due to the peroxidation of the membrane lipids ([40]). It could also be from the formation of Cd-ATPase complexes through SH group of enzyme and increased oxidative stress [19]. HP has the potential to prevent the inhibition of ATPase by Cd as is seen in the results of
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Fig. 5. Histopathological analysis of control, CdCl2 , HP + CdCl2 , and HP groups. Panel A shows brain histopathology of control animals with uniform distribution of neurons. Panel B exhibits brain sections of CdCl2 exposed group showing degenerated neurons along with vacuolated spaces. Panel C shows histology of brain sections of HP pretreated CdCl2 exposed animals where there is only partial neuronal loss with more intact neurons. Panel D depicts HP treated rats resembling normal histology of brain (magnification for all images, 100×).
our study possibly by preventing the peroxidation of the membrane lipids which has also been validated by previous reports [19,37]. Many neurons in the human body secrete acetylcholine at the synapses which acts as neurotransmitters by diffusing across the post-synaptic membranes to transmit the impulse or information. The termination of acetylcholine is carried out by an enzyme called acetylcholinesterase which breaks it down to choline and acetic acid by adding water to the parent biomolecule. Inhibition of this enzyme causes accumulation of acetylcholine which might occur because of free radical production [38] leading to the conditions of cholinergic hyperactivity, convulsion and status epilepticus. The decreased level of AChE in plasma and brain might be one of the indicators for Cd induced hurdle in brain. Natural antioxidants have been found to ameliorate the inhibition of AChE in the case Cd induced neurotoxicity [14,27] and on similar lines HP has been observed to reduce the inhibition of AChE in the brain of rats exposed to Cd. These observations confirm the beliefs that Cd causes toxicity in brain through certain mechanisms which target those biochemical systems which are specifically found in the neurons and cells which inhabit the brain and HP as a high value potential therapeutic agent is up to the task of ameliorating these specialized toxic effects of Cd in the brain. Histological examination of the brain tissue reveals that Cd intoxication caused abnormal structural changes in the brain tissue including apoptosis and vacuolization. With reference to histopathological observation, observable difference has been observed between Cd and HP pre-treated Cd intoxicated rats as there is only partial degeneration of neurons which indicates that HP is capable of preventing the neuronal damage induced by Cd. Therefore, it may be suggested that HP might inhibit Cd induced brain damage.
Conclusion Taking an overview of the results obtained from the study while simultaneously keeping in perspective the background data we can conclude that Cd has various mechanisms to effect toxicity like by peroxidation of lipids, conjugation of thiol groups which would render GSH like antioxidants ineffective and loss of membrane potential and since it has the potential to cross the BBB it is well placed to cause neurotoxicity elucidated by the alteration in neurotoxic markers in the present study but HP has the potential to mitigate the toxic insults of Cd in the brain by varied mechanism involving chelation of Cd, prevention of loss of membrane integrity, protection of thiol containing groups but the underlying property which supplements all these protective actions is its potential as an antioxidant and just like its functional opponent, Cd, it too can cross the BBB and thus HP appears to possess the prospective therapeutic potential which could be harnessed to counter the neurotoxic manifestations of Cd toxicity in the event of an exposure.
Conflict of interests The authors declare that there is no conflict of interests.
Acknowledgment The Grant (no. F. 30-1/2013(SA-II)/RA-2012-14-GE-WES2400), received as Research Award (2012-14) from the University Grants Commission, New Delhi, Government of India to Dr. Suhel Parvez, is thankfully acknowledged.
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