international journal of pharma and bio sciences issn … monniera (brahmi, family:...

Int J Pharm Bio Sci 2014 Oct; 5(4): (B ) 1183 - 1194

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Research Article Biochemistry

International Journal of Pharma and Bio Sciences ISSN

0975-6299

CHRONIC ADMINISTRATION OF METHYLMERCURY ALTERS COGNITIVE

AND MITOCHONDRIAL DYSFUNCTION IN RAT CEREBELLUM – A NOVEL

APPROACH TO THERAPY FOR MINAMATA DISEASE BY A MEDHYA

RASAYANA DRUG BACOPA MONNIERA

JOHNSON CHRISTINAL AND THANGARAJAN SUMATHI*

Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences,

University of Madras,Taramani Campus, Chennai – 600 113, Tamil Nadu, India.

ABSTRACT

Methylmercury (MeHg) is a highly toxic environmental pollutant that causes severe neurological disorders. Considering the neuroprotective property of Bacopa monniera, this study was aimed to evaluate protective effect of Bacopa monniera extract (BME) against MeHg induced mitochondrial dysfunction. Rats were exposed to MeHg by gavage (5mg/kg, b.w) and BME was administered (40 mg/kg, orally) 1 hour before of MeHg for 21 days. After treatment period, MeHg exposure significantly increased the immobility time and decreased learning memory. MeHg exposure resulted in DNA fragmentation and Cytochrome c release in cerebellum of rat. BME pre-treatment reverted all the above changes to near normal. Activities of neurotransmitter enzymes MonoamineOxidase (MAO), Acetyl cholinesterase (AChE), Choline acetyltransferase (ChAT) and Electron Transport Chain (ETC) enzymes were found to be decreased after MeHg exposure. Pre-treatment with BME significantly increased the enzyme activities in cerebellum. The results highlighted that Bacopa monniera can alleviate the MeHg induced mitochondrial dysfunction in rat cerebellum. KEYWORDS: Bacopa monniera, Mitochondrial dysfunction, Methylmercury, DNA fragmentation, Neurotransmitters.

THANGARAJAN SUMATHI

Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical

Sciences, University of Madras,Taramani Campus, Chennai – 600 113, Tamil Nadu, India.



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INTRODUCTION

Methylmercury (MeHg) is one of the most ubiquitous environmental toxins found in high levels in human brains and fish eating wildlife1. MeHg alters the normal structure and function of the central nervous system (CNS), particularly in the cerebral cortex2. The underlying mechanism through which it damages the nervous system is the subject of intensive invivo and invitro research investigations3. Current efforts to elucidate the mechanisms of MeHg toxicity include evaluating their effects on mitochondrial function4, calcium homeostasis perturbation5, the increase of reactive oxygen species (ROS) formation6. The neurological signs observed during intoxication with MeHg compounds, known are man as Hunter’s disease and Minamata disease, including tremor, ataxia, convulsion, erethism and agitation. MeHg accumulated primarily in the spinal dorsal root ganglia, followed by the cerebral cortex and cerebellum. More than 98% of total mercury were in the form of MeHg which was strongly bound to protein7. Thomas and Smith8 reported that MeHg was bound to mainly to glutathione. Decrease in enzymatic activities by MeHg in invivo experiments were also reported 9, 10, 11. A study from our group has shown protective effects of the extract of Bacopa monniera on MeHg induced oxidative stress12 and effect of Bacopa monniera on MeHg induced behavioural and Histopathological changes13. Hence, in this study, we have aimed to extend the work in terms of mitochondrial dysfunctions. Bacopa monniera (Brahmi, family: Scorophulariaceae), a traditional Ayurvedic medicinal plant, is a small creeping annual herb commonly growing in marshy places throughout India ascending to an altitude of 1,320m. Bacopa monniera, also referred to as Bacopa monnieri, Herpestis monniera, water hyssop, and “Brahmi,” has been used in the Ayurvedic system of medicine for centuries. Traditionally, it was used as a brain tonic to enhance memory development, learning, and concentration14. It is extensively used for centuries for treatment of epilepsy, insomnia, and anxiety and also as a mild sedative and memory enhancer15, 16, 17. Several clinical studies have confirmed the beneficial actions of Bacopa monniera18, and the pharmacological

actions are mainly attributed to the saponin compounds present in the alcoholic extract of the plant. The major chemical constituents isolated and characterized from Bacopa are dammarane type of triterpenoid saponins19. Several pharmacological studies20, 21 and clinical studies22, 23 on the extract of Bacopa

monniera standardized to the Bacosides A and B have been reported. The aim of the study was to determine the neuroprotective efficacy of BME against MeHg induced toxicity in mitochondrial dysfunction of cerebellum of rat.

MATERIALS AND METHODS

(i) Chemicals Methylmercury chloride was purchased from Sigma Chemicals. Complex I assay kit was purchased from Sigma Chemicals. Quantikine Rat Cytochrome c release kit was purchased from R&D systems. (ii) Preparation of Bacopa monniera Extract The plant material was collected at Chennai, Tamil Nadu and will be authenticated by Dr. A. Sasikala, Captain Srinivasa Murthi Drug Research Institute for Ayurveda, Arumbakkam, Chennai, Tamil Nadu. The plant was shade dried and coarsely powdered plant material (1KG) was extracted with 90% ethanol in the cold (48 hrs). The extract was filtered and distilled on a cold water bath to obtain a dark green syrupy mass. It was finally dried in vacuum. (iii) Animals Male Wistar Albino rats weighing 250-300g (age of 8-10 weeks) were obtained from Central Animal House, Dr.ALMPGIBMS, University of Madras, Taramani campus, Chennai-113, TamilNadu, India. Rats were housed separately in polypropylene cages and fed a standard pellet diet (purchased from Hindustan Lever) kept under hygienic conditions. Rats were kept on a 12hr light and dark cycles with free access to water (RO water) ad libitium. All experiments and protocols described in the present study were approved by the Institutional Animal Ethics Committee (IEAC) of Dr.ALMPGIBMS, University of Madras, Taramani campus, Chennai-113, TamilNadu, India. Rats were



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divided into four experimental groups of 6 animals each. Group I: control (vehicle orally), Group II:MeHg (5mg/kg, b.w) 24 orally for 21 days, Group III: MeHg+BME (40mg/kg b.w) orally 1 h prior to the administration of MeHg for 21 days, Group IV: BME alone (40mg/kg b.w) orally for 21 days. Experimental animals were handled according to the University and Institutional legislation, regulated by the Committee for the Purpose of Control and Supervision of Experiments on Animals(CPCSEA), Ministry of Environment and Forest ( Animal welfare Division), Government of India (IAEC No. 01/17/2012). (iv) Tissue preparation After the treatment period, experimental animals and control animals were killed by cervical dislocation. Brains were immediately taken out and washed with ice cold saline to remove blood and kept at -80ºC. The cerebellum was rapidly dissected from the intact brain carefully on an ice plate according to the stereotaxic atlas of Paxinos and Watson25. After that mitochondria was isolated according to lee et al 26 for Electron Transport chain enzyme activities. (v) Measurement of mitochondrial ETC

activity (complex I-IV)

The mitochondria were freeze-thawed five times before determination. The activities of complex I (NADH:ubiquinone oxidoreductase) and complex II (Succinate:ubiquinone oxidoreductase) are expressed as nmol/min/mg protein, while complex III (ubiquinol:ferricytochrome c oxidoreductase) and complex IV (cytochrome c oxidase) activities are expressed as k/sec/mg protein. (vi) Determination of Cytochrome c release

from mitochondria. The cerebellum was minced with scissors, and the cytosolic fraction was obtained using a Mitochondrial/Cytosol Fractionation Kit (abcam) according to the manufacturer’s protocol. Cytochrome c released from mitochondria was measured with Quantikine Rat/Mouse Cytochrome c Immunoassay. (vii) Behavioral Parameters All the behavioral parameters were performed at room temperature in a calm room without any outside interference. All of the behavioral

parameters were performed between 10AM and 5PM. Forced Swim Test.The forced swim test was performed according to the method of Porsolt et al27. A vertical glass cylinder (25cm high, 14cm in diameter) was filled with water (30ºC) to a depth of 20 cm. The water depth was adjusted so that the animals must swim or float without their hind limbs or tail touching the bottom. For testing, each animal was placed in the cylinder for 6 min, and the latency to float, and the duration of the floating (i.e. the time during which rat made only the smallest movements necessary to keep their heads above water) was scored. As suggested by Porsolt, only the data scored during the last 4 min were analyzed and presented. In the forced swim procedure, rats are forced to swim in un-escapable situation. After a period of vigorous struggling, the animal becomes immobile, or makes only those movements necessary to keep its head above the water. The immobility observed in this test is considered to reflect a state of despair28. Morris water maze test Spatial learning was assessed using a Morris water maze test. The water maze was a circular plastic pool of 100cm in diameter and filled with water to a depth of 20cm. the water was kept at room temperature (23±1ºC) and was made opaque by adding white paint to prevent the animal from seeing the submerged platform. In the “hidden platform” trials, a round 10-cm diameter platform made of white plexiglass was placed 1 cm below the water surface in the centre of one of the four quadrants. A rat was release into the water at one of the four randomly selected positions near the wall and facing the wall. The latency, defined as the time from release of the rat to climbing on the platform, was recorded. When the rat could not find the platform within 60 sec from the time of release, it was lead to the platform and placed on it for 20 sec before being removed. In such cases, a latency of 60 sec was recorded. Each rat underwent four trials on each of five consecutive days. The pool was fixed at the same position during the experiment. Around the pool were also situated a video device, steel animal racks, and water supply pipes. All were visible from the inside of the pool, and served as distant visible cues for the rat. A visible platform trial, in which rat located the submerged platform by placing a marker on it, was performed after the hidden platform trial was completed.



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(vii) Measurement of neurotransmitter enzyme activity

Activity of Monomine oxidase(MAO), was measured by the flurometric method29. The activities of choline acetyltransferase (ChAT) and acteylcholinesterase(AChE) by the DTNB method30,31. (ix) Analysis of DNA fragmentation DNA extracted from cerebellum of both control and MeHg induced rats were electrophoresed according to the methods described by Rossl 32.

RESULTS

Protective effect of BME on MeHg induced Forced Swim Test Response to inescapable aversive situation interpreted as a measure of depression like behavior .MeHg exposed rats showed significant (p<0.01) longer immobility time than control animals. BME pretreatment was significantly (p<0.05) reduced the immobility. BME alone treated resemble to that of control group (Figure.1). Effect of BME on MeHg induced Morris Water Maze In this study MeHg induced rats showed significant (p<0.01) deficits in learning and memory compared with control rats. Pretreatment with BME was significantly (p<0.05) reduced the deficits. BME alone treated rats showed response similar as a control. (Figure.2). Effect of BME on MeHg induced ETC complex I and complex II MeHg administration caused a significant (p<0.01) decrease in complex I and Complex II activities when compared to control. Whereas, pretreatment with BME significantly (p<0.05) increase the activities. Rats treated with BME

alone did not show any alterations and was similar to that of control (Figure.3) Effect of BME on MeHg induced activities of complex III and complex IV The activity of complex III was found to be significantly decreased (p<0.01) in MeHg induced group, whereas complex IV activity was significantly increased (p<0.01) when compared with control group. Pretreatment with BME significantly (p<0.05) increased the complex III activity and decrease the complex IV activity. BME alone resembles like control (Figure.4). Effect of BME on MeHg induced Cytochrome c release Cytochrome c release is the mark for early stage of apoptosis. In MeHg intoxicated group cytochrome c release was significantly increased (p<0.01). Pretreatment with BME significantly (p<0.05) decreased the release of Cytochrome c. BME alone treated group resembles like control. (Figure.5) Effect of BME on MeHg induced Neurotransmitter enzyme activity The activity of Neurotransmitter enzymes like AChE, ChAT, MAO was significantly decreased (p<0.01) in MeHg induced group. BME pretreatment significantly (p<0.05) increase the activities of neurotransmitter enzymes. BME alone treated group was similar that of control. (Figure.6) Effect of BME on MeHg induced DNA fragmentation DNA fragmentation is a hallmark of apoptosis. Figure.7 shows the effect of BME on MeHg induced DNA fragmentation. DNA ladder was shown in (Lane 5). Lane 2 shows that there was an increased fragmentation observed when compared to control rats (lane 1). BME pretreatment reduced the fragmentation to near normal (lane 3). Lane 5 BME alone treated rats resemble like control.



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FIGURE CAPTIONS

Figure 1 Effect of BME on Forced swim Test of control and experimental rats

Data represents mean ± SD of 6 rats in each group. Values represents the time duration of immobility. *p<0.05 significantly different from control group; **p<0.01 significantly different from MeHg treated group, using one way ANOVA with Tukey’s post hoc test.

Figure 2

Effect of BME on Morris water maze of control and experimental rats

Data represents mean ± SD of 6 rats in each group. Values represent the time to reach platform. *p<0.05 significantly different from control group; **p<0.01 significantly different from MeHg treated group, using one way ANOVA with Tukey’s post hoc test.



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Figure 3 Effect of BME on complex I and complex II of control and experimental rats

Data represents mean ± SD of six rats in each group. Values expressed as nmol/min/mg protein. *p<0.05 significantly different from control group; **p<0.01significantly different from MeHg treated group, using one-way ANOVA with Tukey’s post hoc test.

Figure 4

Effect of BME on complex III and complex IV of control and experimental rats

Data represents mean ± SD of six rats in each group. Values expressed as K/min/mg protein. *p<0.05 significantly different from control group; **p<0.01significantly different from MeHg treated group, using one-way ANOVA with Tukey’s post hoc test.



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Figure 5 Effect of BME on Cytochrome c release of control and experimental rats

Data represents mean ± SD of six rats in each group. Values expressed as ng/mg protein. *p<0.05 significantly different from control group; **p<0.01significantly different from MeHg treated group, using one-way ANOVA with Tukey’s post hoc test.

Figure 6 Effect of BME on neurotransmitter enzymes of control and experimental rats

Data represents mean±SD of six rats in each group. Values expressed as nmol/hr/mg protein. *p<0.05 significantly different from control group; **p<0.01significantly different from MeHg treated group, using one-way ANOVA with Tukey’s post hoc test.



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Figure 7 Effect of BME on DNA fragmentation of control and experimental rats

Figure shows the agarose gel electrophoresis pattern of DNA fragmentation in control and experimental rats. DNA marker was shown in Lane.5. DNA fragmentations were shown in (Lane.2) MeHg intoxicated group. Treatment with BME reduced the fragmentation as shown in Lane.3. In Lane1 (control) there was no fragmentation. Lane.4 (BME alone) similar as control.

DISCUSSION

MeHg induced neurotoxicity is a well described phenomenon. MeHg interferes with such diverse processes as neurotransmitter release and uptake, mitochondrial function, membrane transport, enzyme activity and protein synthesis, there are still no effective treatments available for MeHg poisoning. Our group previously showed the beneficial effects of BME against MeHg induced toxicity12, 13. In those studies BME prevented the neurochemical changes and behavioural changes from MeHg toxicity. In this current study, we have carried out some behavioural parameters and mitochondrial function studies. We studied the antidepressant effect of BME in Forced swim test (FST). In this study MeHg induced rats showed longer immobility time than control rats. The immobility has been expected to reflect a state of “behavioural despair and Variant’33. Initial hypothesis of depression have been formulated about 40

years ago, proposing that the main symptoms of depression are due to functional deficiency of cerebral monaminergic transmitters such as norephineprine (NE), 5-HT and dopamine located at synapses34. Our results are well in accordance with the previous study reported by Nabi et al 35. In our study Bacopa monniera showed the protective effect from immobility induced by MeHg. In earlier studies also this has been proved by Chatterjee et al.36. In Morris water maze experiment MeHg group showed a delay latency to reach the platform. The use of the MWM in assessing learning and memory has been reviewed by Brandies et al 37 and D’Hooge and R, De Deyn38 as has the relationship between performance in the MWM and both neurotransmitter systems and drug effects39. The results obtained from this study are in agreement with Yosidha et al 40, 41, who reported the delay latency to reach the platform induced by MeHg. In our study BME



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showed a memory enhancing effect in Morris water maze. Furthermore, in earlier studies also BME has shown effective memory enhancing property42, 43. Chronic effect of MeHg on DNA fragmentation was studied. In our study DNA was found to be fragmented in MeHg treated group. These findings showed that cleavage of genomic DNA into internucleosome-sized fragments, a hallmark of apoptosis. This effect has been previously reported by Nagshima K44,. BME restored the fragmentation to near normal. In a study, reported by Kamesh and Sumathi45 showed a protective effect of BME on DNA fragmentation induced by HCD induced hypercholesterolemia in rats. Activities of several neurotransmitter enzymes were analyzed in our study. Monoamine oxidase (MAO) serves as an indicator for neurotoxicity46. In Accordance with to Suzuki T47 results, our results also suggested that MAO activity was found to be decreased after exposure of MeHg. Cholinergic neurons are positive markers for the evolution of memory and related disorders affecting acetylcholine and resulting in decreased activity of acetylcholinesterase and choline acetyl transferase48.The activity of AChE and ChAT were also decreased in MeHg exposure group. The result obtained by this study was same as reported previously by Cheng et al 49 and Kobayashi et al 50. BME pretreatment increased the activities of neurotransmitters MAO, AChE, ChAT in cerebellum. Similar studies with reports to BME on neurotransmitter enzymes on cognitive enhancing properties 51, 52 have been reported.We also determined the activity of each complex in the mitochondrial ETC in cerebellum of the rat. Complex I and complex III have been identified as the major generators of Reactive oxygen species (ROS)

in the respiratory chain of mitochondria53,

54Recent studies have shown that complex II was an additional site of ROS generation55. In this study Complex I, II and III activities were decreased in MeHg treated rats. These findings showed MeHg prevent the electron flow and excess of ROS generation. BME restored the activities of ETC enzymes. These results are well in accordance with previous reports by Shinamol et al56 that, 3-nitroprpionic acid induced mitochondrial dysfunction. Cytochrome c release in mitochondria with respect to MeHg toxicity was examined. Cytochrome c release from mitochondria serves as a marker for early and late stage apoptosis57. MeHg induction caused the release of cytochrome c in the cerebellum mitochondria. Our results are well in accordance with previous reports by Mori58 that reported that methylmercury induces cytochrome c release in cerebellum of rat. BME pretreatment reduce the release of cytochrome c release. In conclusion, this study provides substantial evidence to suggest that Bacopa monniera protects the cerebellum from MeHg induced mitochondrial dysfunction. Hence the plat can be useful in the treatment of MeHg induced neurotoxicity.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest

ACKNOWLEDGEMENT

The study was supported by Department of Medical Biochemistry, DR.ALMPGIBMS, University of Madras, Taramani Campus, Chennai-113, Tamil Nadu, India.

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