Role of melatonin against oxidative tissue damage induced by Cleistanthus collinus in rat brain

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1 Indian J Med Res 3, October 29, pp Role of melatonin against oxidative tissue damage induced by Cleistanthus collinus in rat brain M. Jayanthi, R. Raveendran & Debdatta Basu * Departments of Pharmacology & * Pathology, Jawaharlal Institute of Postgraduate Medical Education & Research, Puducherry, India Received September, 28 Background & objectives: The leaves of Cleistanthus collinus, an extremely poisonous plant are consumed for suicidal purposes in various parts of India. The mortality rate is high and there is no antidote. In this study, we attempted to delineate oxidative stress as a possible mechanism of action of C. collinus toxicity in rats and the role of melatonin against injury to brain and heart caused by C. collinus. Methods: Adult Wistar rats (3-2 g, n = 6 per group) of either sex were used. C. collinus at 8 mg/kg body weight (LD ) was administered orally followed by melatonin mg/kg body weight ip or cysteine mg/kg body weight ip (standard) after 2 h. Malondialdehyde (MDA), reduced glutathione (GSH), glutathione peroxidase and catalase levels in brain, heart and blood were estimated and histopathological examinations (brain and heart) were done. For the survival study, rats were treated with increasing doses of melatonin (, and mg/kg body weight ip) following a lethal dose of C. collinus (. g/kg body weight orally). Results: The results showed a significant (P<.) increase in blood and brain MDA levels and decrease in tissue GSH in the LD group. This was accompanied by marked gliosis, spongiform necrosis and lymphocytic inflammatory infiltrates in brain and marked congestion, inflammation and muscle necrosis in heart. Melatonin significantly (P<.) reduced lipid peroxidation and reversed the histopathological changes induced by C. collinus in the brain but not in the heart. Interpretation & conclusion: Our results suggest that oxidative mechanisms play an important role in C. collinus induced tissue damage and melatonin, by balancing oxidant-antioxidant status ameliorates oxidative organ injury in brain due to C. collinus toxicity. Key words Antioxidant - Cleistanthus collinus - melatonin - morphological changes - oxidative stress Cleistanthus collinus (Roxb.) Benth. and Hook f. (Euphorbiaceae) is a poisonous plant growing on dry hills in various parts of India. Its leaves are consumed for suicidal purposes, associated with a high mortality due to lack of an antidote. C. collinus poisoning is reported to be associated with life threatening neuromuscular and cardiac as well as acid-base disturbances 2-4. Drowsiness, decreased muscle power, respiratory failure and uncontrolled hypotension have been reported suggesting a central nervous system toxicity as well. However, the exact mechanism of toxicity is not known and there is no 467

2 468 INDIAN J MED RES, october 29 suitable antidote that can be used clinically in cases of poisoning with this highly toxic plant. Diphyllin glycosides, cleistanthin A and ceistanthin B have been found to be the toxic constituents in the leaf extract 6,7. Cleistanthins A and B were found to reduce the viability of cells and produce DNA strand breaks in in vitro studies 8-. Oxygen radical induced injury to cells is considered to be an important factor responsible for C. collinus induced toxicity. Acute lethal doses of C. collinus leaf extract given to rats and rabbits have been shown to reduce glutathione levels in various tissues 2. Cysteine has been shown to reduce mortality in the rats administered with C. collinus leaf extract 3. Hence it was hypothesized that an effective antioxidant could reduce the mortality and morbidity and as a result it would improve the longevity. Melatonin is an effective antioxidant and free radical scavenger. It has shown cytoprotective action against many highly toxic agents like paraquat (herbicide) and carbon tetrachloride that produce cell damage 4. Melatonin was shown to be more effective than vitamins E and C in reducing molecular damage that occurs under high free radical generating conditions. Its unique intracellular distribution and cascade of scavenging actions brought about by its metabolites also contribute to its high efficacy. Many studies on animals have demonstrated the antioxidant effects of melatonin 6-8. The present study was undertaken to investigate the role of oxidative stress in the heart and brain of C. collinus intoxicated rats. It was also proposed to study the potential role of exogenous melatonin in reversing the oxidative damage and thereby prolonging the survival of rats that are administered a lethal dose of C. collinus. Material & Methods Preparation of plant extract: C. collinus plant was identified at field areas of Karasoor near Puducherry (India) and the leaves were collected in the month of May. The specimen was authenticated by Dr Pragasam A, Head of the Department of Botany, K.M. Centre for P.G. Studies, Puducherry. A voucher specimen (V. no. JIP 234/6) with botanist authentication was kept for reference in the departmental library of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry. The leaves were air dried in the shade and powdered using a mixer. A total of 87 g of the leaf powder was added to distilled water ( g in 2 ml), boiled, filtered and evaporated using heating mantle to obtain an aqueous extract of 72. g. The yield was 8.46 per cent. Animals and experimental design: Healthy adult Wistar albino rats of either sex weighing 3-2 g were obtained from the Central Animal House, JIPMER, Puducherry. The study was approved by The Institute Animal Ethics Committee. The animals were maintained in standard laboratory conditions (2 h : 2 h dark and light cycle and 2 ± 2 C temperature) and provided standard food and water ad libitum. The rats were fasted 24 h prior to the study with free access only to water. The animals were divided into seven groups of six animals each for the survival study and into five groups of six animals each for the biochemical and histopathological study. Survival study: Group : received 2-3 ml of distilled water orally. Group 2: received 2-3 ml of distilled water orally and after 2 h, mg/kg body weight melatonin (Sigma, USA) ip repeated every 24 h for 3 days. Group 3: received. g/kg of C. collinus mixed with distilled water orally. Group 4: received a lethal dose of C. collinus at. g/kg orally and after 2 h, mg/kg of melatonin ip. Melatonin in the same dose was repeated every 24 h for 3 days if the rat survived. Group : received C. collinus at. g/kg orally and after 2 h, mg/kg of melatonin ip. Melatonin in the same dose was repeated every 24 h for 3 days if the rat survived. Group 6: received C. collinus at. g/kg orally and after 2 h, mg/kg of melatonin ip. Melatonin in the same dose was repeated every 24 h for 3 days if the rat survived. Group 7: received C. collinus at. g/kg orally and after 2 h, mg/kg cysteine ip. Cysteine in the same dose was repeated every 24 h for 3 days if the rat survived. After treatment, the rats were housed in separate cages for each group. The survival time of each rat was noted and recorded as the time taken for death of the animal from the time of administration of C. collinus. Biochemical and histopathological study: Group : received 2-3 ml of distilled water orally.

3 Jayanthi et al: Melatonin for C. collinus poisoning 469 Group 2: received 2-3 ml of distilled water orally and after 2 h, mg/kg of melatonin ip. Melatonin in the same dose was repeated every 24 h for 3 days. Group 3: received 8 g/kg of C. collinus mixed with distilled water orally. Group 4: received 8 g/kg of C. collinus and after 2 h, mg/kg of melatonin ip. Melatonin in the same dose was repeated every 24 h for 3 days if the rat survived. Group : received 8 g/kg of C. collinus and after 2 h, mg/kg of cysteine ip. Cysteine in the same dose was repeated every 24 h for 3 days if the rat survived. The LD (lethal dose) and the LD of C. collinus were selected for survival and biochemical and histopathological study respectively as obtained from previous studies 2,9. Melatonin used at doses of 4 and mg/kg ip has shown to have protective effect against drug induced myocardial injury in previous studies 2, 2. In this study the dose response relationship was evaluated with an additional dose of mg/kg. Collection of blood and tissues : After 6 h of C. collinus administration, 4 ml blood was withdrawn from all the rats in heparinised tubes by cardiac puncture under ether anaesthesia. Then the rats in the test and control groups were sacrificed by cervical decapitation and their heart and brain were excised. Processing of tissues for histopathological studies: One half of heart and one half of brain were fixed in 4 and 4 per cent formalin respectively. The fixed tissues were processed, embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin (H & E) and observed under microscope (Olympus, Japan). The pathologist was blinded to the treatment. The brain sections were examined for gliosis, spongiosis, inflammatory infiltrate, oedema and meningeal changes. The heart sections were examined for inflammatory changes or muscle necrosis. The findings were classified subjectively, depending upon the extent of damage into grades of increasing severity as mild, moderate and marked changes while compared to those in a normal rat. Processing of tissues for biochemical studies: The other half of the heart and brain tissue was immediately immersed in cold phosphate buffer, ph 8. It was blotted free of blood and tissue fluids and then weighed on electronic balance (Sartorius, Germany). The tissues were chopped into small pieces and homogenized in ice-cold phosphate buffer (ph 8) at a concentration of per cent (w/v). This would release soluble protein leaving only membrane and nonvascular matter in a sedimental form. It was then centrifuged in cooling centrifuge (Hettich Zentrifugen, Germany) at 62 g for min. The supernatant was separated and further centrifuged at 4 g for 4 min at 4º C. The final clear supernatant was stored at -2º C for evaluation of reduced glutathione (GSH) and malondialdehyde (MDA). Processing of blood: Blood samples were collected in heparinised tubes and plasma was separated by centrifugation and stored at -2º C for estimation of MDA levels. The RBCs were washed three times with cold saline and centrifuged after each wash for min at 4ºC. It was diluted with cold distilled water ( in ) and stored at -2ºC for evaluation of haemoglobin, GSH, catalase and glutathione peroxidase (GP x ) within h using spectrophotometer (Hitachi, Japan). Malondialdehyde and glutathione assays: Plasma and tissue samples of brain and heart were assayed for products of lipid peroxidation 22. Results were expressed as nmol MDA/ml plasma or nmol MDA/g tissue. GSH was determined by the spectrophotometric method using Ellman s reagent 23. The amount of glutathione in RBC was expressed as μg of GSH/g Hb and that in the tissues as μg of GSH/g weight of wet tissue. Catalase and glutathione peroxidase assays: Catalase and glutathione peroxidise activity in the RBCs were determined by spectrophotometric method 24,2. Catalase activity was expressed as k/ml, k being the rate constant in the reaction where catalase converts hydrogen peroxide to water and oxygen. Glutathione peroxidise activity was expressed as units of GPx/g haemoglobin where one unit of glutathione peroxidise activity is defined as the change in log concentration of GSH (μ moles) in unit time. Statistical analysis: Survival data were expressed as number of hours after administration of C. collinus. Kaplan Meier survival analysis with log rank test was carried out to analyze the survival data using SPSS version 3 software (LEAD Technologies, Inc., USA). Biochemical data were expressed as mean ± SEM. One-way ANOVA with Dunnett s post test was used to analyze the biochemical parameters with Graph Pad, Instat Version 3.6, USA. Non parametric ANOVA with Kruskal-Wallis test was used to analyze the grades of histopathological changes in brain and heart tissues. P<. was considered statistically significant.

4 47 INDIAN J MED RES, october 29 Results A significant (P<.) rise in plasma and brain MDA levels could be seen in the rats that received C. collinus alone. The rise was not significant in the heart. There was a significant (P<.) reversal of MDA levels in brain with melatonin and cysteine administration. No significant reduction of MDA levels were noted in plasma and heart on treatment with melatonin or cysteine (Fig. ). There was a significant (P<. & P<.) reduction in GSH levels in RBCs of rats that received C. collinus alone compared to both vehicle control and the melatonin control groups. This reduction was not significant in brain and heart tissue. Both cysteine and melatonin failed to raise GSH concentrations in RBC and brain. GSH levels in heart were neither affected by the toxin nor by the drugs (Fig. 2). A significant (P<.) reduction was observed in GP x and catalase levels of C. collinus administered rats. Melatonin and cysteine did not raise the GP x concentrations significantly, though cysteine reversed the changes in catalase levels significantly (P<.) (Table I). The brain of rats that received C. collinus alone showed marked gliosis, spongiform necrosis and lymphocytic inflammatory infiltrates as against normal architecture shown by the brains of vehicle and melatonin Fig.. Effect of melatonin and cysteine on malondialdehyde (MDA) concentrations in plasma, brain and heart of rats administered C. collinus. Data are expressed as mean ± SEM. a P <. compared to vehicle control group, b P<. compared to melatonin control group, c P<. compared to C. collinus group. C. collinus was given at a dose of 8 g/kg body weight po, melatonin at mg/kg body weight ip and cysteine at mg/kg body weight ip, n=6 in each group. VC, vehicle control; MC, melatonin control; CC, C. collinus; mel; melatonin; cyst, cysteine. Fig 2. Effect of melatonin and cysteine on reduced glutathione (GSH) levels in RBC, brain and heart of rats administered C. collinus. Data are expressed as mean ± SEM. a P<. compared to vehicle control, b P<. compared to melatonin control. C. collinus was given at a dose of 8 g/kg body weight po, melatonin at mg/kg body weight ip and cysteine at mg/kg body weight ip, n=6 in each group. VC, vehicle control, MC, melatonin control; CC, C. collinus; mel, melatonin; cyst- cysteine. Table I. Effect of melatonin and cysteine on glutathione peroxidase and catalase levels in RBCs of rats administered C. collinus Groups Glutathione Catalase (k/ml) peroxidise (U/g Hb) Vehicle control ± ±. Melatonin control ± ±. C. collinus ± 6.9 a,b 6.3 ±.9 a,b C. collinus + melatonin 4.63 ± ±.82 C. collinus + cysteine ± ±.6 c Values are mean ± SEM, n=6 in each group a P<. compared to vehicle control b P<. compared to melatonin control c P<. compared to C. collinus group C. collinus was given at a dose of 8 g/kg body weight oral, melatonin was given at a dose of mg/kg body weight ip and cysteine at a dose of mg/kg body weight ip control rats (Fig. 3). On treatment with melatonin, there was a significant reduction in these changes but not with cysteine (Table II). The hearts of the rats that received C. collinus alone showed marked congestion, inflammation and muscle necrosis while those in vehicle and melatonin control groups showed a normal architecture (Fig. 4). Melatonin or cysteine could not reduce these changes significantly (Table II). Lethal dose of C. collinus significantly reduced the survival time of rats (P<.; One-way ANOVA) compared to vehicle control and melatonin control rats which survived beyond 72 h. The differences in survival between the control and the drug treated groups were not statistically significant by log rank test (Fig. ).

5 Jayanthi et al: Melatonin for C. collinus poisoning 47 Fig. 3. Histopathological changes in rat brain (H& E stain 2x) (a) vehicle control showing normal architecture (b) melatonin control showing normal architecture (c) following C. collinus administration showing spongiform necrosis and lymphocytic inflammatory changes (d) following C. collinus administration and treatment with melatonin showing near normal architecture (e) following C. collinus administration and treatment with cysteine showing mild spongiosis. Table II. Histopathological changes in brain and heart of rats administered C. collinus Groups No. of rats showing No. of rats showing histopathological histopathological changes in the brain changes in the heart Discussion We have previously seen prominent oedema, spongiform necrosis and dilated venous spaces in the brain of rats administered C. collinus (unpublished data). There are no published data that describe Vehicle control Melatonin control C. collinus a C. collinus + Melatoninb C. collinus + cysteine Mild Moderate Marked Mild Moderate Marked P<. compared to vehicle control and melatonin control group in heart and brain; b P<. compared to C. collinus group for brain only; C. collinus was given at a dose of 8 g/kg body weight po, melatonin at mg/kg body weight ip and cysteine at mg/kg body weight ip a

6 472 INDIAN J MED RES, october 29 Fig. 4. Histopathological changes in rat heart (H& E stain 2x) (a) vehicle control rat showing normal architecture (b) melatonin control rat showing normal architecture (c) following C. collinus administration showing myocardial necrosis and mononuclear inflammatory infiltrates (d) following C. collinus administration and melatonin treatment showing mild congestion. (e) following C. collinus administration and cysteine treatment showing mild inflammatory changes. histopathological changes in brain and heart following C. collinus poisoning. Melatonin treated rats showed near normal brain morphology but this was not so in the heart suggesting that treatment with potent antioxidants like melatonin may protect the brain from significant morphological damage but not the heart. There was a significant rise in the blood and brain lipid peroxide (MDA) concentrations after acute administration of C. collinus indicating oxidative stress and resultant brain tissue damage. Treatment of rats with the melatonin and cysteine seemed to afford protection against this damage by significantly reducing MDA concentrations in brain. This is comparable to the protective effect demonstrated by melatonin in inhibiting oxidative damage to brain induced by many toxic substances and also in a variety of neurological disease models26. Melatonin and its metabolites are known to readily cross blood-brain barrier and capable of neutralizing reactive oxygen species and reactive nitrogen species27.

7 Jayanthi et al: Melatonin for C. collinus poisoning 473 Fig.. Kaplan Meier survival curve showing time to death (h) for rats that received C. collinus alone, C. collinus followed by melatonin at, and mg/kg b.wt. and cysteine at mg/kg b.wt. The differences in survival are not statistically significant (log rank analysis). In the present study, GSH concentration in blood was significantly reduced which is also indicative of oxidative stress. However, GSH concentrations in brain and heart were not significantly reduced. This finding is different from that of Sarathchandra et al 2 who reported that C. collinus induced significant reduction of glutathione concentrations in heart and brain tissues of rats. However, they sacrificed rats 2 h after C. collinus administration which could have led to the depletion of GSH stores and thus a decrease in its concentration before the compensatory mechanisms for oxidative stress could have set in. In our study, the insignificant change in the glutathione concentrations in brain and heart tissues at the end of 6 h after C. collinus administration may be attributed to the endogenous compensatory mechanisms that got stimulated to combat oxidative stress which would have masked the actual reduction in GSH concentrations by C. collinus. Melatonin and cysteine were not able to significantly raise the blood and tissue GSH concentrations. This could be due to the fact that GSH is utilized by the oxidative reactions taking place in the tissues or it could have been bound by the toxin. One of the postulates suggested earlier was binding of the toxin to sulphydryl group enzymes and leading to their inhibition and thus affecting GSH concentrations 3. The reduction seen in blood catalase and glutathione peroxidase concentrations following toxin administration further confirmed the presence of oxidative stress. In our study, melatonin and cysteine were found to reduce oxidative brain damage, though significant reversal was obtained only with melatonin. Both did not show a protective effect in the heart because oxidative stress was not pronounced in the heart. Melatonin being a lipid soluble agent, is capable of detoxifying radicals in the lipid rich environments of the cell, i.e., cellular membranes. Besides it is also capable of entering the aqueous environments of the cell and protects cytosolic molecules and nuclear DNA from free radical damage 4. Melatonin was earlier known to act on ML and ML 2 receptors which were thought to be involved in the regulation of retinal function, circadian rhythms and reproduction 28. Melatonin was also reported to produce hypotension by acting on ML receptors in rat brain 29. These functions cannot explain its protective role in our study. Recently, there are reports of melatonin acting on MT and MT 2 G-protein linked membrane receptors and nuclear RZR/ROR receptors to regulate gene transcription and antioxidant enzyme levels 3,3. Upregulation of antioxidant enzyme levels by acting on MT /2 and the nuclear receptors is a possibility that cannot be ruled out. In the survival study, both melatonin and cysteine did not significantly prolong the survival of rats administered C. collinus. This is comparable to the findings of an earlier study showing only a per cent reduction in mortality of rats 9. In another survival study by Jose et al 32, potassium channel modulators (minoxidil and glibenclamide) could not prolong the survival of C. collinus treated rats. From the results, it can be concluded that C. collinus has induced brain damage due to oxidative stress and melatonin by its antioxidant effects could prevent the brain injury. There was neither a significant reduction in GSH nor a rise in the MDA levels in heart, but cardiac tissue damage was found. Hence it is postulated that mechanisms other than oxidative stress may play a role in the cardiac damage like a direct action of the toxin on enzymes/proteins as reported earlier 33. Considering that there is no antidote or specific treatment, melatonin may be supplemented in the treatment of patients with C. collinus poisoning along with other supportive measures. Acknowledgment The first author gratefully acknowledges the intramural fund received from the Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry to carry out the work. Conflicts of interest: None.

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