MELATONIN AND RANITIDINE IN COMBINATION PROTECTS AGAINST PIROXICAM-INDUCED GASTRIC ULCERATION IN RATS THROUGH ANTIOXIDANT MECHANISM(S)

Transcription

1 Journal of Cell and Tissue Research Vol. 13(3) (213) (Available online at ISSN: ; E-ISSN: Original Article MELATONIN AND RANITIDINE IN COMBINATION PROTECTS AGAINST PIROXICAM-INDUCED GASTRIC ULCERATION IN RATS THROUGH ANTIOXIDANT MECHANISM(S) BASU, A. 1, GHOSH, A. K., 1 MITRA, E., 1 BASU, M., 1 MUKHERJEE, D., 1 DUTTA, M., 1,2 PATTARI, S. K., 3 ROY, S. S., 4 CHATTOPADHYAY, A., 2 AND BANDYOPADHYAY, D. 1#? 1 Oxidative Stress and Free Radical Biology Laboratory, Department of Physiology, University of Calcutta, University College of Science and Technology, 92 APC Road, Kolkata 7 9; # Principal Investigator, Centre with Potential for Excellence in a Particular Area (CPEPA), University of Calcutta, 92, APC Road, Kolkata 7 99; 2 Department of Physiology, Vidyasagar College, 39 Sankar Ghosh Lane, Kolkata 7 6; 3 R. N. Tagore International Institute of Cardiac Sciences, Mukundapur, Kolkata 7 99; 4 Molecular Endocrinology Division, Indian Institute of Chemical Biology, 4-Raja S. C. Mullick Road, Kolkata 7 32 ^( Presently - National Centre for Cell Science, Pune E. mail: debasish63@gmail.com, Cell: Received: October 5, 213; November 1, 213 Abstract: The anti-ulcer effect of different doses of melatonin and ranitidine was studied, in separate experiments, against piroxicam-induced gastric ulceration in rats, with the intent of determining the minimum dose of each of the drugs at which neither of them were effective individually. Melatonin and ranitidine, both exert protection to the gastric mucosa individually, dose-dependently, by decreasing the piroxicam-induced oxidative stress in the gastric tissue. Our studies revealed that melatonin and ranitidine both at a dose of 2 mg/kg BW i.p., individually, could not protect the gastric tissue from being ulcerated. However, when rats were pre-treated with melatonin and ranitidine in combination, at the doses at which neither was effective individually, the combined therapy completely prevented ulcers following piroxicam treatment. Melatonin and ranitidine in combination ameliorated the oxidative stress induced in the gastric tissue due to piroxicam by preventing the alterations in the bio-markers of oxidative stress and the activities and levels of antioxidant enzymes. The biochemical analyses were supported by macroscopic and microscopic studies of gastric tissue. Thus, the combination seems to work synergistically to reduce piroxicam-induced gastric damage in rats, strongly indicating that it may be a safe gastro-protective co-therapy especially, in situations where the use of antiinflammatory drugs is the only choice. Key words: Antioxidant, Gastric ulceration, Melatonin, Oxidative stress, 3779

2 J. Cell Tissue Research INTRODUCTION Melatonin was first isolated from bovine pineal glands and was structurally identified in 1958 by Aaron Lerner [1]. It is a highly evolutionarily conserved pineal secretory molecule which is chemically known as N-acetyl-5-methoxytryptamine, present in virtually all organisms like microbes, plants and animals including humans [2-5]. Melatonin is known as the hormone of darkness, secreted in darkness in both dayactive (diurnal) and night-active (nocturnal) animals [6]. Many biological effects of melatonin are produced through activation of melatonin receptors [7]. The well-documented effects of melatonin and its metabolites as antioxidants have shown that they protect cells, tissues and organs from oxidative damage induced by ROS as well as from nitrogenbased reactants [8, 9]. Melatonin is particularly effective in neutralizing the hydroxyl radical ( OH) which attacks DNA, proteins and lipids leading to a variety of disorders [1, 11]. Melatonin also detoxifies superoxide anion free radical (O2 -) [12], nitric oxide (NO ), peroxynitrite anion (ONOO-) [13], hypochlorous acid (HOCl) [14], the haemoglobin oxoferryl radical [15], ABTS + cation radical and possibly the peroxyl radicals (LOO ) [16], all of which cause cell damage [17]. In addition, melatonin inhibits inducible nitric oxide synthetase (inos) [18] and stimulates several antioxidant enzymes [19]. Additionally, it increases the efficiency of the electron transport chain and, as a consequence, likely reduces electron leakage and the generation of free radicals [2]. Due to its antioxidative actions, melatonin protects against heavy metals [21] and other toxic agents and it works synergistically with exercise to improve stroke volume [22]. anesthesia. It is very commonly used in treatment of peptic ulcer disease (PUD) and gastro esophageal reflux disease (GERD) [26]. Ranitidine at a high dose, appears to decrease mucosal perfusion in patients with acute renal or cardiac failure and increases their risk of death [27]. All drugs in its class decrease gastric intrinsic factor secretion which can significantly reduce absorption of protein-bound vitamin B12 in humans [28]. Elderly patients taking H2 receptor antagonists are more likely to require vitamin B12 supplementation than those not taking such drugs [29]. H2 blockers may also reduce the absorption of drugs (azole antifungals, calcium carbonate) that require an acidic stomach. Antacid preparations such as ranitidine by suppressing acid mediated break down of proteins, leads to an elevated risk of developing food or drug allergies. This happens due to undigested proteins then passing into the gastrointestinal tract where sensitisation occurs. It is unclear whether this risk occurs with only long or short-terms uses as well [3]. A previous work by Bandyopadhyay et al (22) [63] demonstrated that co-administration of melatonin in combination with ranitidine or omeprazole prevents the stomach from undergoing ulceration in the face of restraint cold stress [31]. Herein, we provide evidence that melatonin and ranitidine in combination provide complete protection to the rat gastric mucosa against piroxicam-induced ulceration at the doses at which neither of these drugs alone has the capability to provide protection, individually. The results further demonstrate that this combination exerts its protective effects through antioxidant mechanism(s). METHODS AND MATERIALS Melatonin protects the gastric mucosa from oxidative damage caused by ROS in different experimental ulcer models [23-25]. Use of non-steroidal antiinflammatory drugs (NSAIDs), due to various reasons, has been reported to be increased globally [61-63]. Bandyopadhyay et al. [33] has demonstrated, perhaps for the first time, the protective effects of melatonin against piroxicam-induced gastric ulceration in rat model. Ranitidine is a histamine H 2 -receptor antagonist that inhibits stomach acid production. Ranitidine is used sometimes to treat upper gastrointestinal bleeding and to prevent stress ulcers, stomach damage from use of NSAIDs, and aspiration of stomach acid during 378 Drugs, reagents and antibodies: Melatonin, thiobarbituric acid (TBA), eosin, NAD+, 2,2-dithiobisnitro benzoic acid (DTNB), xanthine, xanthine oxidase, cytochrome c, fast blue BB salt, nitro blue tetrazolium (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and glutathione peroxidase kit were obtained from Sigma (St. Louis, MO, USA). Hematoxylin, H 2 O 2, dimethyl sulfoxide (DMSO) and piroxicam were obtained from Merck Limited (Delhi, India). The superoxide dismutase (SOD) 1(C-17), SOD 2(G-2) and actin (I-19) antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Monoclonal anti-catalase was obtained from Sigma. Other reagents used were of analytical grade.

3 Basu et al. Animals: Male Wister rats, weighing 18-2 g, were obtained from the CPCSEA approved local animal supplier (Chakraborty Enterprise). The animals were first acclimatized to laboratory conditions and their handling and the experiments were carried out as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) [IAEC/PROPOSAL/DB-4/ 21], Ministry of Environment, Government of India. All the experimental protocols had the approval of the Institutional Animal Ethics Committee (IAEC) of Department of Physiology, University of Calcutta. Animal treatments - Piroxicam-induced gastric ulceration and dose-dependent protection by melatonin or ranitidine as well as protection by melatonin and ranitidine in combination: In the first set of experiments, dose-response studies for both melatonin and ranitidine were carried out separately. Fasted male Wister rats (water ad libitum), were divided into five groups in both of the cases (i.e., melatonin or ranitidine dose-response experiments). The rats of the first group constituted the vehicle treated control. The rats of the second group were fed orally with NSAID of choice, i.e., piroxicam (3 mg/kg body weight [bw]). In case of ranitidine dose-response experiment, the rats of the third, fourth and fifth group were injected intraperitoneally with different doses of ranitidine (i.e., 2, 4 and 6 mg/kg bw, respectively) 3 min prior to piroxicam treatment. In case of melatonin doseresponse experiment, the rats of the 3 rd, 4 th and 5 th group were injected intraperitoneally with different doses of melatonin (i.e., 2, 4 and 6 mg/kg bw, respectively) 3 min prior to piroxicam treatment. In the second set of experiments, fasted male Wister rats (water ad libitum) weighing gm, were divided into five groups. The rats of the first group constituted the vehicle treated control. The rats of the second group were fed orally with NSAID of choice, i.e., piroxicam (3 mg/kg bw). Rats of the third group were injected intraperitoneally with ranitidine (2 mg/kg bw) 45 min prior to piroxicam treatment. The rats of the fourth group were also injected intraperitoneally with melatonin (2 mg/kg bw) 3 min prior to piroxicam treatment. The fifth group were injected intraperitoneally with both ranitidine (2 mg/kg bw body weight) and melatonin (2 mg/kg bw), 45 and 3 minutes before piroxicam treatment, respectively. In both set of experiments,the animals were kept at room temperature and were sacrificed 4 hrs after piroxicam treatment by cervical dislocation following mild ether anesthesia to assess the degree of gastric lesion. The stomach tissues were collected after surgically opening the abdominal cavity and after proper processing were stored at -2 C for further biochemical analyses. Measurement of mean ulcer Index: The grades of lesions were scored according to the following scale:, no pathology; 1, small 1 2mm ulcers; 2, medium 3 4 mm ulcers; 4, large 5-6 mm ulcers; 8, ulcers >6 mm. The sum of the total ulcer score in each group of rats, divided by the number of animals, was expressed as the mean ulcer index [31]. Measurement of lipid peroxidation, protein carbonyl, reduced GSH and total sulfhydryl content: A portion of the fundic stomach was homogenized (5%) in ice-cold.9% saline (ph 7.) with a Potter Elvenjem glass homogenizer (Belco Glass Inc., Vineland, NJ, USA) for 3 s and lipid peroxides in the homogenate were determined as thiobarbituric acid reactive substances (TBARS) according to the method of Buege and Aust [32] with some modification as described by Bandyopadhyay et al. [33]. Protein carbonyl content was estimated by DNPH assay as adopted by Levine et al. [34]. The level of GSH in the gastric tissue (as acid soluble sulfhydryl) was estimated by its reaction with DTNB (Ellman s reagent) following the method of Sedlack and Lindsay [35] with some modifications [33]. Total sulfhydryl group was measured following the method as described by Sedlack Lindsay [35] Measurement of the activities of gastric peroxidase (GPO), superoxide dismutase (SOD), and catalase: A portion of the fundic stomach was homogenized (1% homogenate) in.25 M sucrose and 5 mm phosphate buffer (ph 7.2) and the mitochondrial fraction was prepared. The GPO activity in this mitochondrial fraction was measured using iodide as an electron donor. The enzyme activity was expressed as units/mg protein [31]. Copper-zinc superoxide dismutase (SOD1) activity was measured by hematoxylin autooxidation method of Martin et al. [36] with some modifications [37]. Manganese superoxide dismutase (SOD2) activity was measured in the mitochondrial fraction by the xanthine oxidase cytochrome c method as described by McCord and Fridovich [38] with some modifications as adopted by Bandyopadhyay et al. [31]. The enzyme activity was expressed as U/mg 3781

4 J. Cell Tissue Research Mean Ulcer Index Fi C P PM2 PM4 PM6 D Mean Ulcer Index C P PR2 PR4 PR6 A Figs. 1 (A - D): Dose-dependent protection by melatonin (M) and ranitidine (R) respectively against piroxicam-induced gastric mucosal ulceration (mean ulcer index). (B), (E): The macroscopic view of mucosal surface of whole stomach showing protective effect of melatonin and ranitidine respectively against piroxicam-induced (P) damage of rat gastric tissue. (C), (F): Representative images of hematoxylineosin stained sections of stomach tissue (magnification 1X, 2X and 4X) (for the clarity of presentation, histological figures are shown at three different magnifications.). Rats were treated with piroxicam (P) (fed orally) and increasing doses of melatonin (PM) (i.p.) and ranitidine (PR) (i.p.) respectively. Control (C) animals were treated similarly with vehicle only. Values are mean ± S.E.M of six rats in each group; p <.1 versus C. p <.1 versus piroxicam-treated animals using ANOVA. 3782

5 Basu et al. protein. Catalase was assayed by the method of Beers and Sizer [39] with some modifications as adopted by Chattopadhyay et al. [4]. The enzyme activity was expressed as µm H 2 O 2 consumed / min /mg protein. Measurement of the activities of glutathione reductase, glutathione peroxidase (GPx) and glutathione-s-transferase (GST): The glutathione reductase was carried out according to the method of Krohne-Ehrich et al. [41]. The specific activity of the enzyme was calculated as units/min/mg tissue protein. The glutathione peroxidase activity was measured according to the method of Paglia and Valentine (1967) [42] with some modifications as adopted by Chattopadhyay et al. [4]. The specific activity was expressed as nmol of NADPH produced/ min/mg tissue protein. The glutathione-s-transferase activity of the rat gastric tissue was measured sphectrophotometrically according to Habig et al. [43]. The enzyme activity was expressed as units/ min/mg of tissue protein. Elvenjem glass homogenizer (Belco Glass Inc., Vineland, NJ, USA) for 3s. The homogenate was then centrifuged at 5 g for 1 min and the supernatant was again centrifuged at 12, g for 15 min to obtain the mitochondria. The mitochondrial pellet thus obtained was resuspended in the buffer and used for measuring the mitochondrial enzymes. Measurement of pyruvate dehydrogenase activity: Pyruvate dehydrogenase activity was measured spectrophotometrically according to the method of Chretien et al. [46] with some modifications by following the reduction of NAD+ to NADH at 34nm. Values were expressed as Units/mg protein. Measurement of isocitrate dehydrogenase activity: Mitochondrial isocitrate dehydrogenase activity was measured according to the method of Duncan et al [47] by measuring the reduction of NAD+ to NADH at 34nm with the help of a UV- VIS spectrophotometer. The enzyme activity was expressed as Units/mg protein. Measurement of tissue hydroxyl radical ( OH) generated in vivo: The OH generated in the stomach tissue in vivo was measured by using dimethyl sulfoxide (DMSO) as a specific OH scavenger following the method of Bandyopadhyay et al. [33]. Dimethyl sulfoxide forms a stable product (methane sulfonic acid [MSA]) on reaction with OH. Accumulation of MSA was measured to estimate the OH generated after forming a colored complex with Fast Blue BB salt. The values obtained were expressed as nm of OH/g of stomach tissue. Measurement of Xanthine oxidase/xanthine dehydrogenase activity for the assessment of superoxide anion radical (O2 - ) in vivo; Xanthine oxidase (XO) was assayed by measuring the conversion of xanthine to uric acid following the method of Greenlee and Handler [44] with some modifications [37]. The enzyme activity was expressed as milli Units/ mg protein. Xanthine dehydrogenase (XDH) was assayed by following the reduction of NAD + to NADH according to the method of Strittmatter [45] with modifications [37]. The enzyme activity was expressed as milli Units/mg protein. Measurement of the activities of pyruvate dehydrogenase and some of the key mitochondrial Kreb s cycle enzymes: A portion of the gastric tissue was homogenized (1%) in icecold 5 mm phosphate buffer, ph 7.4, with a Potter 3783 Measurement of α-ketoglutarate dehydrogenase activity: Alpha-ketoglutarate dehydrogenase activity was measured spectrophotometrically according to the method of Duncan et al. [47]. The enzyme activity was expressed as Units/mg protein. Measurement of succinate dehydrogenase activity: Mitochondrial succinate dehydrogenase activity was measured spectrophotometrically by following the reduction of potassium ferricyanide (K 3 FeCN 6 ) at 42nm according to the method of Veeger et al [48] with some modifications. The enzyme activity was expressed as Units/mg protein. Measurement of activities of some of the mitochondrial respiratory chain enzymes; NADH-Cytochrome c oxidoreductase activity was measured spectrophotometrically by following the reduction of oxidized cytochrome c at 565 nm according to the method of Goyal and Srivastava [49]. The activity of the enzyme was expressed as units/ min/mg tissue protein. The cytochrome c oxidase activity was determined spectrophotometrically by following the oxidation of reduced cytochrome c at 55 nm according to the method of Goyal and Srivastava [49]. The enzyme activity was expressed as units/min/mg tissue protein. Measurement of the activity of MMP2 and MMP9 by Gelatin Zymography: The fundic part

6 J. Cell Tissue Research 1 8 A Mean Ulcer Index Fig. 2: (A): Protective effect of the combination (2mg/kg bw, i.p. each) of melatonin (M) and ranitidine (R) against piroxicam-induced gastric mucosal ulceration (mean ulcer index). (B): The macroscopic view of mucosal surface of whole stomach showing protective effect of melatonin and ranitidine combination against piroxicam-induced (P) damage of rat gastric tissue. (C): Representative images of hematoxylin-eosin stained sections of stomach tissue (magnification 1X, 2X and 4X) (for the clarity of presentation, histological figures are shown at three different magnifications.). Rats were treated with piroxicam (3mg/kg bw, fed orally) (P), piroxicam (3mg/kg bw, fed orally) + ranitidine (2mg/kg bw, i.p.) (PR), piroxicam (3mg/kg bw, fed orally) + melatonin (2mg/kg bw, i.p.) (PM) and piroxicam (3mg/kg bw, fed orally) + ranitidine (2mg/kg bw, i.p.) + melatonin (2mg/kg bw, i.p.) (PRM). Control (C) animals were treated similarly with vehicle only. Values are mean ± S.E.M of six rats in each group; p <.1 versus C. p <.1 versus piroxicam-treated animals using ANOVA. 3784

7 Basu et al. of the gastric mucosa was suspended in phosphatebuffered saline containing protease inhibitors, minced, and incubated for 1 min at 4 C. After centrifugation at 12,xg for 15 min, the supernatant was discarded. The pellet was extracted in the lysis buffer (1 mm Tris-HCl ph 8., 15 mm NaCl, 1% Triton X-1 plus protease inhibitors) and centrifuged at 12,xg for 15 min. Tissue extracts were preserved at -7 C and used in future studies. MMP activity was significantly decreased within a short period of time if the mucosa was processed in the absence of protease inhibitors. For assay of MMP-2 and MMP- 9 activity, mucosal extracts were electrophoresed in SDS-polyacrylamide gel containing 1 mg ml -1 gelatin under nonreducing conditions. The gels were washed in 2.5% Triton-X-1 and incubated in CAB buffer (4 mm Tris-HCl, ph 7.4,.2 M NaCl, 1 mm CaCl2) for 18 h at 37 C and stained with.1% Coomassie Blue followed by destaining [18]. The zones of gelatinolytic activity came as negative staining [5]. Measurement of the level of antioxidant enzymes through Western blot analysis: Western blot analysis was performed with the stomach homogenates which were prepared as described earlier by Bandyopadhyay et al. [33] with minor modifications [37]. The pixel density of bands obtained through Western blotting was quantified using Image J software (NIH, Bethesda, MD, USA). Estimation of proteins: Proteins of the different samples were determined by the method of Lowry et al. [52]. Histological studies: Following sacrifice of rats, the stomach was surgically extirpated from each group and opened through vertical incision along the greater curvature and photographs were immediately taken of the inside surface of the stomach. The stomach tissues were then washed in.9% saline and kept in 1% buffered formalin for histopathological studies. The tissues were processed, sections obtained and then hematoxylin and eosin, periodic acid-schiff and alcian blue staining was done for the observation of possible histopathological changes like congestion, hemorrhage etc. [53] and changes in the mucin pattern of the gastric tissue, if any. The tissue sections were examined under an Olympus BX51 (Olympus Corporation, Tokyo, Japan) microscope and images were captured with a digital camera attached to it. Quantification by confocal microscopy: Stomach sections (5 μm thick) were stained with Sirius red (Direct Red 8; Sigma Chemical Co, Louis, MO, USA) according to the method of Roy et al. [54] and imaged with laser scanning confocal system (Zeiss LSM 51 META, Germany). and the stacked images through multiple slices were captured. The digitized images were then analyzed using image analysis system (Image J, NIH Software, Bethesda, MI) and the total collagen area fraction of each image was measured and expressed as the % collagen volume. Statistical evaluation: Each experiment was repeated at least three times with different rats. Data are presented as means ± S.E.M. Significance of mean values of different parameters between the treatment groups were analyzed using one way analysis of variances (ANOVA) after ascertaining the homogeneity of variances between the treatments. Pairwise comparisons were done by calculating the least significance. Statistical tests were performed using Microcal Origin version 7. for Windows. RESULTS Figures 1(A to C and D to F) demonstrates that both melatonin and ranitidine, in separate experiments, has dose-dependently protected the piroxicam-induced gastric ulceration in rats as measured through mean ulcer index (A and D). At the dose of 2mg / kg bw injected i.p., both melatonin or ranitidine alone could not protect against piroxicam-induced gastric ulceration. However, melatonin or ranitidine alone was found to almost completely protect against piroxicaminduced gastric ulceration at the dose of 6 mg /kg bw, injected i.p. The macroscopic observation reveals hemorrhagic ulcer spots over the surface of the gastric mucosa with marked mucosal congestion and hemorrhage (Fig.1 B,E). The hemorrhage was also noticed on rat stomach surface when they were pretreated with melatonin at the dose of 2mg / kg bw, injected,i.p. However, no hemorrhage was observed on the stomach mucosal surface when the rats were pre-treated with melatonin or ranitidine at the doses of 4 or 6mg /kg bw, injected i.p. No mucosal congestion or hemorrhage was noticed on the stomach surface of the control rats. The microscopic examination carried out through H and E stained stomach tissue sections from piroxicam 3785

8 J. Cell Tissue Research Figs. 3: (A), (B): Dose-dependent protection by melatonin (M) and ranitidine (R) respectively against piroxicam-induced alteration in the lipid peroxidation level in rat gastric mucosa. Rats were treated with piroxicam (P) (fed orally) and increasing doses of melatonin (PM) (i.p.) and ranitidine (PR) (i.p.) respectively. Control (C) animals were treated similarly with vehicle only. Values are mean ± S.E.M of six rats in each group; p <.1 versus C. p <.1 versus piroxicam-treated animals using ANOVA. LPO [n moles of TBARS/ mg of A C P PM2 PM4 PM6 LPO [n moles TBARS/ mg B C P PR2 PR4 PR6 Figs.4: (A), (B): Dose-dependent protection by melatonin (M) and ranitidine (R) respectively against piroxicam-induced alteration in the reduced glutathione content in rat gastric mucosa. GSH [n moles of GSH/mg of proteins] A C P PM2 PM4 PM6 GSH [n moles GSH/ mg B C P PR2 PR4 PR6 Animal Groups Figs.5: (A), (B): Dose-dependent protection by melatonin (M) and ranitidine (R) respectively against piroxicam-induced alteration in the Cu-Zn superoxide dismutase activity in rat gastric mucosa. Cu-Zn SOD [units/ mg A C P PM2 PM4 PM6 Cu-Zn SOD [units/mg of B C P PR2 PR4 PR6 treated rats showed marked congestion of mucosal and submucosal vessels along with haemorrhage (Fig.1C,F). Mucosal vascular congestion and haemorrhage was also noticed in the stomach tissue sections of rats pre-treated with melatonin or ranitidine at the dose of 2mg /kg bw, injected i.p. However, the severity was less compared to piroxicam-treated rats (Fig.1C,F]). Mild mucosal congestion was seen in the rats pre-treated with melatonin or ranitidine at the dose of 4mg /kg bw, i.p. However, no congestion or haemorrhages were observed in the stomach tissue sections from the rats pre-treated with melatonin or ranitidine at the dose of 6 mg /kg bw, i.p.(fig.1 C,F) The tissue sections from the control rats did not show any mucosal congestion or haemorrhage (Fig. 1C,F ). Figure 2 demonstrates that pre-treatment of rats with melatonin and ranitidine in combination at the dose of 2mg / kg bw, i.p., completely protected the stomach from being ulcerated due to piroxicam, as revealed from mean ulcer index (Fig. 2A) and macroscopic observation of the surface of the 3786

9 Basu et al. stomach mucosa (Fig. 2B). At this dose neither melatonin nor ranitidine alone could provide protection against piroxicam-induced gastric ulceration in rat. Microscopic examination of the stomach tissue sections, stained with H and E, from rats pre-treated with melatonin or ranitidine at the dose of 2mg /kg bw, injected i.p., showed marked mucosal vascular congestion and haemorrhage, however, with lesser severity than the rats treated with piroxicam alone. However, when the rats were pre-treated with the combination of melatonin and ranitidine (each, at the dose 2 mg /kg bw, injected i.p.) no haemorrhage was noticed although marked vascular congestion persisted (Fig.2 C). Figure 3 (A and B) demonstrates piroxicam-induced elevation (56.25%) in the level of lipid peroxidation (LPO) of the rat gastric tissue compared to control. Pre-treatment of rats with melatonin or ranitidine at increasing doses was found to provide dosedependent protection against piroxicam-induced elevation in the level of LPO in the rat stomach tissue. Melatonin or ranitidine, at the dose of 6mg /kg bw, injected i.p., was found to completely protect the level of LPO from being increased. A significant decrease (43.81%) in the level of GSH was observed following treatment of rats with the present dose of piroxicam, compared to control. A dose-dependent protection of the gastric tissue level of GSH, however, was observed when the rats were pre-treated with melatonin or ranitidine (Figs. 4 A, B). Here also, the tissue GSH level was found to be almost completely protected from being decreased by melatonin or ranitidine at the dose of 6 mg/ kg bw, injected i.p. Treatment of rats with piroxicam at the indicated dose was found to significantly increase (53.48%) the activity of Cu-Zn SOD compared to control, an important antioxidant enzyme of the gastric tissue. Melatonin or ranitidine, at increasing doses, was found to protect the activity of this enzyme from being increased. Melatonin or ranitidine, at the dose of 6mg /kg bw, injected i.p., was found to protect the activity of Cu-Zn SOD almost completely (Figs. 5 A,B). Figure 6 (A,B) depicts a significant increase in the activity of catalase (34.78%) compared to control), another important antioxidant enzyme of the rat gastric tissue. Pre-treatment of rats with melatonin or ranitidine was found to protect, dose-dependently, the activity of catalase from being increased. At the dose of 6 mg /kg bw, injected i.p., melatonin or ranitidine was found to almost completely protect the activity of catalase from being increased. The results presented in figures 7 (A,B,C,D) demonstrate that rats when pre-treated with a combination of melatonin and ranitidine (each at a dose of 2mg /kg bw, injected i.p.) almost completely protected the levels of LPO, protein oxidation (measured as protein carbonyl content), GSH and total thiol (TSH) of the rat gastric tissue from being altered. Melatonin or ranitidine alone, at the dose of 2 mg/kg bw, injected i.p., however, were found to be unable to provide protection against piroxicaminduced alterations in the parameters. Treatment of rats with piroxicam at the indicated dose was found to cause significant elevation in the activities (Figs. 8 A,C) and the levels of enzyme proteins (Figs.8 B,D) of both Cu-Zn SOD (55.82%) and Mn-SOD (activity; 58.95%, protein level; 18.14%) of rat gastric tissue, compared to control. When the rats were pre-treated with the combination of melatonin and ranitidine (each at the dose of 2 mg /kg bw, injected i.p.), the activity as well as the protein levels of both the enzymes were found to be almost completely protected from being altered. Melatonin or ranitidine alone (at the dose of 2mg / kg bw, injected i.p.) was found to be unable to provide protection against piroxicam-induced alteration in the activity and the protein level of both the enzymes. The activity and the protein level of rat gastric catalase were found to be significantly increased 34.78%) following treatment of rats with piroxicam, compared to control. However, when the rats were pre-treated with melatonin and ranitidine in combination (each at the dose of 2mg /kg bw, injected i.p.), the activity as well as the protein level (31.16% compared to control) of catalase were found to be almost completely protected from being increased (Figs. 9 A and B). Melatonin or ranitidine alone, however, failed to provide any protection against piroxicam-induced alteration in the activity and the protein level of rat gastric catalase. Figure 1 demonstrates a significant decrease (43.91%) in the activity of gastric peroxidise, compared to control, one of the important antioxidant enzyme of rat gastric mucosa, following treatment of rats with the present dose of piroxicam. The 3787

10 J. Cell Tissue Research Figs. 6: (A), (B): Dose-dependent protection by melatonin (M) and ranitidine (R) respectively against piroxicam-induced alteration in the catalase activity in rat gastric mucosa. Catalase [m mol H2O2 consumed/ min/ mg C P PM2 PM4 PM6 A Catalase [m moles of H 2O2 consumed/ mg proteins] C P PR2 PR4 PR6 B Figs. 7: (A - D): Protective effect of the combination (2mg/kg bw, i.p. each) of melatonin (M) and ranitidine (R) against piroxicam-induced changes in the biomarker of oxidative stress in rat gastric mucosa. (A): lipid peroxidation level, (B): protein carbonyl content, (C): reduced glutathione content, (D): total sulfhydryl group content. LPO [n moles TBARS/ mg A Protein Carbonyl content [n mol/ mg B GSH [n moles GSH/ mg C TSH [n moles TSH/ mg D activity of this enzyme was found to be completely protected from being decreased when the rats were pre-treated with melatonin and ranitidine in combination (each at a dose of 2mg /kg bw, injected i.p.). However, melatonin or ranitidine alone, failed to provide any protection to the activity of GPO from being decreased. Treatment of rats with piroxicam, at the present dose, caused significant decrease in the activities of GPx (29.92%), GR (42.12%) and GST (54.74%), compared to control, as well as a decrease in the protein level of GST (38.5%), also compared to control (Figure 11 A to D). The activities of the glutathione metabolizing enzymes and the protein level of GST were found to be significantly protected from being decreased when the rats were pre-treated with melatonin and ranitidine in combination (each at the dose of 2mg /kg bw, injected i.p.). Melatonin or ranitidine alone failed to provide any significant protection to the activities of these enzymes and the GST protein level. Figure 12 A demonstrates that treatment of rats with piroxicam significantly decreased the gastric free mucin content. Pre-treatment of rats with melatonin and ranitidine in combination (each at the dose of 2mg /kg bw, injected i.p.) almost completely 3788

11 Basu et al. Figs. 8 A-D: Protective effect of the combination (2mg/kg bw, i.p. each) of melatonin (M) and ranitidine (R) against piroxicam-induced changes in the activities and enzyme protein levels of Cu- Zn superoxide dismutase and Mn-superoxide dismutase in rat gastric mucosa. (A): Cu-Zn superoxide dismutase activity, (B): Cu-Zn superoxide dismutase enzyme protein level, (C): Mn-superoxide dismutase activity, (D): Mn-superoxide dismutase enzyme protein level. Cu-Zn SOD [units/mg of Cu-Zn SOD [ Pixel Density (Arbitrary Unit)] A- B Mn SOD [units/mg C Mn SOD [ Pixel Density (Arbitrary Unit)] D Figs. 9 A,B: Protective effect of the combination (2mg/kg bw, i.p. each) of melatonin (M) and ranitidine (R) against piroxicam-induced changes in the activity and enzyme protein level of catalase in rat gastric mucosa. (A): catalase activity, (B): catalase enzyme protein level. Catalase [m moles of H2O2 consumed/ mg proteins] A Catalase [ Pixel Density (Arbitrary Unit)] B Fig. 1: Protective effect of the combination (2mg/kg bw, i.p. each) of melatonin (M) and ranitidine (R) against piroxicam-induced changes in the activity of an important gastric antioxidant enzyme gastric peroxidase in rat gastric mucosa. GPO [units/ mg protected the gastric free mucin from being decreased. Melatonin or ranitidine alone failed to provide any protection to gastric free mucin. Figure 12B shows the Alcian blue stained rat gastric tissue sections which reveals increased Alcian blue positive cells in piroxicam treated as well as melatonin or ranitidine protected (each at the dose of 2 mg /kg bw, injected i.p.) indicating increased secretion of acid mucin. However, when the rats were pre-treated with melatonin and ranitidine in combination (each at the dose of 2mg /kg bw, injected i.p.) the number of Alcian blue positive cells were decreased indicating significant protection against piroxicam-induced gastric lesion. Similarly, Fig. 12 C demonstrates that 3789

12 J. Cell Tissue Research the tissue sections from piroxicam-treated as well as melatonin (2 mg / kg bw, injected i.p.) or ranitidine (2mg /kg bw, injected i.p.) protected animals decreased staining intensity with Periodic acid-schiff stain indicating decreased secretion of neutral mucin in the rat stomach. However, when the rats were pre-treated with melatonin and ranitidine in combination (each at the dose of 2mg /kg bw, injected i.p.), the gastric tissue was found to be significantly protected as secretion of neutral mucin increased. The changes in the tissue sections were demonstrated at three magnifications for a better understanding. Treatment of rats with piroxicam caused depletion of gastric tissue collagen, as was studied through Acid Sirius staining of the tissue sections and quantified through confocal laser scanning microscopy, which could not be protected from being depleted when the rats were pre-treated with melatonin or ranitidine at the dose of 2 mg /kg bw, injected i.p. However, when the rats were pre-treated with melatonin and ranitidine in combination (each at the dose of 2 mg /kg bw, injected i.p.) the gastric tissue collagen was significantly protected from being decreased. The percent collagen area volume was determined through Image J Software of NIH, USA (Figs.13 A, B and C [(i) and (ii)]). We have studied the levels of Pro MMP-9, Pro MMP-2 and MMP 2 through zymography. Figure 14 A reveals that treatment of rats with piroxicam caused elevation of the level of Pro-MMP-9 and decreased the levels of Pro MMP-2 and MMP-2. However, when the rats were pre-treated with melatonin and ranitidine in combination (each at the dose of 2 mg /kg bw, injected i.p.) the levels of Pro MMP-9, Pro MMP-2 and MMP-2 were almost completely protected from being altered. Melatonin or ranitidine alone, at the dose of 2mg /kg bw, injected i.p., failed to provide any protection to Pro MMP-9, Pro MMP-2 and MMP-2. Table 1 reveals that treatment of rats with piroxicam increased the in vivo generation of hydroxyl radical several fold compared to control as well as caused increased activity of xanthine oxidase (44.98%) and xanthine dehydrogenase (3.79%), total enzyme and their ratio, all compared to control. However, when the rats were pre-treated with melatonin and ranitidine in combination (each at the dose of 2mg /kg bw, injected i.p.), the level of hydroxyl radical was almost completely prevented from being increased and the activities of the proxidant enzymes responsible for the generation of superoxide anion free radicals were also found to be significantly protected from being increased. Table 1 further reveals that treatment of rats with piroxicam significantly decreased the activities of PDH (8.4%), ICDH (79.93%), α- KGDH (83.19%) and SDH (51.41%), all Kreb s cycle enzymes as well as two of the respiratory chain enzymes, like NADH-Cyt c reductase (49.6%) and Cytochrome oxidase (46.67%), compared to respective controls. The activities of these enzymes were found to be significantly protected from being decreased when the rats were pre-treated with melatonin and ranitidine in combination (each at the dose of 2mg/kg bw, injected i.p.). However, melatonin or ranitidine alone were found to be unable to protect these important enzymes from being decreased. DISCUSSION Gastric ulceration has become a general health problem of global concern [55,56]. Uses of antiinflammatory drugs of synthetic origin accordingly, are also rising world-wide. Misuse or over-use of the anti-inflammatory drugs is posing additional problems to clinicians and scientists in the management of such patients as well. The principal problem with these anti-inflammatory drugs is that apart from causing inhibition to cyclo-oxygenase 2 (COX-2), they also bring about inhibition of cyclooxygenase 1 (COX-1) which produces gastro-friendly prostaglandins. Till date, none of the COX-inhibitors are perfect COX-2 inhibitors. The COX-2 mediates inflammatory prostaglandins at the sites of inflammation. This means whatever be the type of COX-inhibitors used there remain always a chance of these affecting the gastric physiology with compromised mucosal integrity and with the resultant gastric ulcerations and in many of the cases, deadlier perforating ulcers of the stomach which in many cases are fatal. Use of anti-inflammatory drugs, particularly, non-steroidal anti-inflammatory drugs (NSAIDs), is also associated with the development of oxidative stress in the gastric tissue and consequential ulceration of the gastric mucosa [33]. perhaps in other tissues as well. Gastrointestinal bleeding is also related to the type of NSAIDs and the dosage [57]. Piroxicam, a classic non-selective, COX-1 preferent NSAID, is widely 379

13 Basu et al. Figs.11: Protective effect of the combination (2mg/kg bw, i.p. each) of melatonin (M) and ranitidine (R) against piroxicam-induced changes in the activities of glutathione peroxidase, glutathione reductase and glutathione S transferase and the enzyme protein level of glutathione S transferase in rat gastric mucosa. (A): glutathione peroxidase activity, (B): glutathione reductase activity, (C): glutathione S transferase activity, (D): glutathione S transferase enzyme protein level. GPx [n mols of NADPH produced/min/mg of A GR [units/min/mg of B GST [units/min/mg of C GST [ Pixel Density (Arbitrary Unit)] D used by patients requiring anti-inflammatory intervention [58]. Piroxicam is a member of the oxicam group of NSAIDs. According to the Biopharmaceutic Drug Classification System (BCS) proposed by Amidon et al. [59], piroxicam is a class 2 drug with low solubility and high permeability. Its pharmacokinetic pattern is characterized by slow and gradual absorption via the oral route and a long halflife of elimination, rendering a prolonged therapeutic action. Piroxicam like other NSAIDs, is a nonselective COX inhibitor possessing both analgesic and antipyretic properties. It undergoes enterohepatic circulation [6]. There are a number of drugs to control gastric hyperacidity and ulceration of the mucosa. However, these drugs are not devoid of side effects, and may be harmful in many ways if used for a longer period of time [33,61,62]. Therefore, search is on to find out a drug or a drug combination which possess anti-secretary, anti-ulcer and antioxidant properties. The primary objective of this work is to address the question of whether we can protect the gastric mucosa against piroxicaminduced gastric ulceration in rats by melatonin and ranitidine in combination at the lowest possible dose, because both melatonin and ranitidine are now well established as an anti-ulcer agent. Although melatonin, even at high pharmacological dose is well tolerated by human, and referred to as a non-toxic molecule, ranitidine, however, has number of side-effects, if used for longer periods of time or at situations where its use is the only choice [26-3]. It was shown earlier by Bandyopadhyay et al. [31] that the side-effects of ranitidine can be minimized, when its long-term use is the only choice, if co-administered with melatonin. Results presented in figures 3-6 demonstrate clearly that both melatonin and ranitidine are capable of providing protection to the gastric mucosa against piroxicam-induced gastric injury in a dose-dependent manner. In most of the experiments, the piroxicaminduced gastric ulceration was maximally protected by melatonin or ranitidine at 6 mg/kg bw (i.p.). The biochemical changes observed have been confirmed by the mean ulcer index study and the macroscopic and microscopic observations of the gastric mucosa (Figs. 1A-1F) which indicate gastric tissue damage following piroxicam treatment. This gastric mucosal injury was found to be protected when the rats were pre-treated with different doses of either melatonin or ranitidine. The figures 1 and 3-6 further reveals 3791

14 J. Cell Tissue Research C B Free Mucin content [mg/g of tissue] A Fig. 12: Protective effect of the combination (2mg/kg bw, i.p. each) of melatonin (M) and ranitidine (R) against piroxicam-induced alteration in the stomach mucin content. (A): quantitative analysis of gastric free mucin content, (B): Alcian blue staining for the gastric mucin (magnification 1X, 2X and 4X) (for the clarity of presentation, histological figures are shown at three different magnifications.), (C): periodic acid-schiff staining of gastric mucosa. Rats were treated with piroxicam (3mg/kg bw, fed orally) (P), piroxicam (3mg/kg bw, fed orally) + ranitidine (2mg/kg bw, i.p.) (PR), piroxicam (3mg/kg bw, fed orally) + melatonin (2mg/kg bw, i.p.) (PM) and piroxicam (3mg/kg bw, fed orally) + ranitidine (2mg/kg bw, i.p.) + melatonin (2mg/kg bw, i.p.) (PRM). Control (C) animals were treated with vehicle only. Values are mean ± S.E.M of six rats in each group; p <.1 versus C. p <.1 versus piroxicam-treated animals using ANOVA. 3792

15 Basu et al. that melatonin or rantidine individually, at a dose of 2 mg/kg bw, i.p., does not protect against piroxicaminduced gastric ulceration. This prompted us to investigate whether these two ineffective doses of melatonin and rantidine would protect the gastric mucosa when administered in combination. Our experiments revealed that pretreatment of rats with the combined doses of melatonin and rantidine almost completely inhibited the gastric mucosal ulceration as measured by mean ulcer index and with clear supportive macroscopic and microscopic observations (Figs. 2A-C) indicating the strong potential of this combination. The individual doses of melatonin or ranitidine, however, were found to be ineffective. Additionally, when the rats were treated singly with melatonin or rantidine, they had no effect on the gastric mucosa (positive control). Earlier studies by Bandyopadhyay et al. [31] have demonstrated the co-therapeutic effect of melatonin with ranitidine or omeprazole in providing protection to the gastric mucosa against cold-restraint stressinduced gastric ulceration at the doses at which none of them were effective individually [31]. The results of the present investigation indicate clearly that this combination not only inhibited piroxicaminduced ulceration of the gastric mucosa but also prevented the piroxicam-induced oxidative stress as evident from the significantly reduced level of LPO and protein carbonyl and increased level of gastric GSH and TSH (Figs. 7A-D) which indicates that this combination of melatonin and rantidine are highly efficient in mitigating the ill effects of piroxicam administration on gastric mucosa. Melatonin and rantidine in combination, at their otherwise ineffective doses, also prevents the alterations in the activities of key gastric antioxidant enzymes like Cu-Zn SOD, Mn SOD (Figs. 8 A,C), catalase (Figs. 9A), gastric peroxidise (Fig. 1), glutathione peroxidise, glutathione reductase, glutathione S transferase (Figs.. 11 A-C). The alteration in the activities of the gastric antioxidant enzymes, like, Cu-Zn SOD, Mn SOD (Figs.. 8 B,D), catalase (Figs. 9B) and glutathione S transferase (Figs. 11D) following piroxicam treatment were further analysed through western blot analysis. It was revealed that the expressions of those enzymes were increased significantly following piroxicam treatment. However, pre-treatment of rats with melatonin and ranitidine in combination at the dose at which they are ineffective individually, protected the activities as well as the levels of the enzyme proteins from being altered indicating again the superior efficacy of this combination in giving protection to the gastric mucosa against piroxicaminduced alterations. However, neither melatonin nor ranitidine had any effect on the antioxidant levels and the activities of antioxidant enzymes indicating that they do not influence these parameters by themselves (positive control). It was shown earlier [63] that piroxicam-induced gastric ulceration results in vivo generation of OH. Our experiments again confirmed the generation of OH in vivo following treatment of rats with the indicated dose of piroxicam. However, when the rats were pre-treated with the combination of ranitidine and melatonin, the formation of OH was prevented from being increased. However, melatonin or ranitidine alone, at their otherwise individual ineffective dose, failed to provide any protection against OH formation indicating that this combination has strong potential to either inhibit or quench the OH that is generated in vivo following piroxicam treatment [table 1]. Treatment of rats with piroxicam was also found to be involved with the generation of O 2, another ROS, as is evident from the enhanced activities of xanthine oxidase (XO) and xanthine dehydrogenase (XDH) as well as the increased XO/XDH ratio and the level of total enzyme (XO+XDH) [table 1]. These indicate strongly that piroxicam-induced gastric ulceration is the outcome of severe oxidative stress developed within the gastric tissue of rats and in vivo generation of ROS may be associated with the gastric mucosal injury. Pre-treatment of the rats with the combination of melatonin and ranitidine, at their otherwise ineffective doses, almost completely protected the activities of these enzymes from being elevated. This indicates that the combination may have the potential to either inhibit the generation of O 2 or scavenge this ROS. Mitochondria are the seat as well as the principal target of oxidative stress. In our studies, the effect of piroxicam on the mitochondrial enzymes related to energy metabolism was studied. The current studies revealed the status of activity of pyruvate dehydrogenase (PDH) and some of the mitochondrial Kreb s cycle enzymes, particularly, ICDH, alpha- KGDH, and SDH related to ATP production in mitochondria. In each case, the activities of the 3793

16 J. Cell Tissue Research Percentage (%) area of collagen i Percentage (%) area of collage C ii Figs. 13: Protective effect of the combination (2mg/kg bw, i.p. each) of melatonin (M) and ranitidine (R) against piroxicam-induced gastric ulceration. (A): Representative images of acid-sirius stained sections of stomach tissue (magnification 1X, 2X and 4X) (for the clarity of presentation, histological figures are shown at three different magnifications.), (B): Images captured by confocal laser scanning microscope for quantification of fibrosis (magnification 2X and 4X), (C): Histogram showing % collagen volume in the gastric tissues (i-for 2X magnification and ii-for 4X magnification). Rats were treated with piroxicam (3mg/kg bw, fed orally) (P), piroxicam (3mg/kg bw, fed orally) + ranitidine (2mg/kg bw, i.p.) (PR), piroxicam (3mg/kg bw, fed orally) + melatonin (2mg/kg bw, i.p.) (PM) and piroxicam (3mg/kg bw, fed orally) + ranitidine (2mg/kg bw, i.p.) + melatonin (2mg/kg bw, i.p.) (PRM). Control (C) animals were treated with vehicle only. Values are mean ± S.E.M of six rats in each group; p <.1 versus C. p <.1 versus piroxicam-treated animals using ANOVA. 3794

17 Basu et al. Figs. 14.: Protective effect of the combination (2mg/kg bw, i.p. each) of melatonin (M) and ranitidine (R) against piroxicam-induced alteration associated with MMP-9 and MMP-2 of rat gastric mucosa. (A): gelatine zymography showing the alterations in pro MMP-9, pro MMP-2 and MMP-2, (i): pro MMP-9 activity, (ii): pro MMP-2 activity, (iii): MMP-2 activity. Rats were treated with piroxicam (3mg/kg bw, fed orally) (P), piroxicam (3mg/kg bw, fed orally) + ranitidine (2mg/kg bw, i.p.) (PR), piroxicam (3mg/ kg bw, fed orally) + melatonin (2mg/kg bw, i.p.) (PM) and piroxicam (3mg/kg bw, fed orally) + ranitidine (2mg/kg bw, i.p.) + melatonin (2mg/kg bw, i.p.) (PRM). Control (C) animals were treated with vehicle only. Values are mean ± S.E.M of six rats in each group; p <.1 versus C. p <.1 versus piroxicam-treated animals using ANOVA. 14A pro MMP 9[Pixel Density (Arbitrary Units)] i pro MMP 2 [Pixel Density(Arbitrary Units)] ii MMP 2[Pixel Density (Arbitrary Units)] iii Table 1: Piroxicam induced gastric ulceration and protection by the combination of ranitidine and melatonin in rats.. Each value is a mean ± S.E.M. of six samples in each group. Statistical analysis done by one-way ANOVA followed by Scheffe multiple comparison tests. α p <.1 is the p value vs. control,. α1 p <.1 is the p value vs. piroxicam-treated animals, β NS vs. piroxicam-treated animals, γ p <.5 vs. piroxicam-treated animals. NS, not significant. Parameters for ROS generation Mitochondrial Kerb s cycle enzymes Mitochondrial respiratory chain enzymes Experiments Control Piroxicam [P] OHº generated [nmoles /gm tissue] XO [milli units /mg XDH [milli units /mg XO+XDH [milli units /mg Piroxicam + Piroxicam + Ranitidine [PR] ± ± 1.15α ± 1.15β 1.26 ± ±.28α 2.22 ±.8β 2.27 ± ±.25α 3.19 ±.1γ 3.54 ± ±.45α 5.41 ±.9γ XO/XDH ratio.55 ±.12.7 ±.9α1.69 ±.2β PDH [units /mg ICDH [units /mg α- KGDH [units/ mg SDH [units/mg NADH-Cytochrome c oxidoreductase [units /mg Cytochrome c oxidase [units /mg ± ±.167α 3.9 ±.137β 5.98 ± ±.2α 1.27 ±.27β 2.38 ±.7.4 ±.21α.54 ±.25β ± ±.268α ±.15β 9.48 ± ±.67α 4.72 ±.71β.75 ±.39.4 ±.45α.43 ±.28β Melatonin [PM] ± 1.15γ 2.18 ±.22γ 3.18 ±.9γ 5.36 ±.26γ.68 ±.7β 3.22 ±.116β 1.3 ±.13β.57 ±.22γ ±.7β 4.89 ±.35β.48 ±.36γ Piroxicam + Ranitidine + Melatonin [PRM] ± 1.15α ±.16α1 2.3 ±.13α ±.23α1.56 ±.7α ±.16α ±.63α ±.3α ±.667α1 9.7 ±.77α1.77 ±.48α1 3795