Toxicology Mechanisms and Methods

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The Effect of Etoricoxib, a Cyclooxygenase-2-Specific Inhibitor, on the 1,2-Dimethylhydrazine-Administered Rat Intestinal Membrane Structure and Function Neha Mittal a; Shailender Singh Kanwar a; Sankar Nath Sanyal a a Department of Biophysics, Panjab University, Chandigarh, India

Online Publication Date: 01 January 2008 To cite this Article: Mittal, Neha, Kanwar, Shailender Singh and Sanyal, Sankar Nath (2008) 'The Effect of Etoricoxib, a Cyclooxygenase-2-Specific Inhibitor, on the 1,2-Dimethylhydrazine-Administered Rat Intestinal Membrane Structure and Function', Toxicology Mechanisms and Methods, 18:1, 53 - 62 To link to this article: DOI: 10.1080/15376510701380372 URL: http://dx.doi.org/10.1080/15376510701380372

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Toxicology Mechanisms and Methods, 18:53–62, 2008 c Informa Healthcare USA, Inc. Copyright ISSN: 1537-6516 print; 1537-6524 online DOI: 10.1080/15376510701380372

The Effect of Etoricoxib, a Cyclooxygenase-2–Specific Inhibitor, on the 1,2-Dimethylhydrazine–Administered Rat Intestinal Membrane Structure and Function Neha Mittal, Shailender Singh Kanwar, and Sankar Nath Sanyal Department of Biophysics, Panjab University, Chandigarh, India

ABSTRACT To gain insight into the chemopreventive effects of etoricoxib, which is a selective inhibitor of cycloxygenase-2, a study was carried out in the procarcinogen 1,2-dimethylhydrazine–treated rat intestine. The male Sprague Dawley rats were divided into three different groups. Group 1 served as control (vehicle treated). All animals in Group 2 were given a weekly subcutaneous injection of 1,2-dimethylhydrazine (DMH; 30 mg/kg body weight) for 6 weeks. Group 3 animals were given an additional oral dose of etoricoxib (6 mg/kg body weight) along with weekly DMH injections for 6 weeks. At the end of 6 weeks of treatments, the results indicated significant alterations in the biochemical parameters, membrane lipid composition, and membrane fluorescence studies of the intestine in the presence of DMH, which were recovered nearly to the control level and, therefore, may suggest the chemopreventive efficacy of etoricoxib against the experimental intestinal cancer in rats. KEYWORDS Antioxidative Defense System; 1,2-Dimethylhydrazine; Etoricoxib; Membrane Fluorescence; Membrane Lipids; Rat Intestine

INTRODUCTION

Received 31 January 2007; accepted 8 March 2007. Address correspondence to Dr. S. N. Sanyal, Professor, Department of Biophysics, Punjab University, Chandigarh-160 014, India. E-mail: [email protected]

Etoricoxib is a recent entry into the field of cycloxygenase-2 (COX-2) selective inhibitors called the nonsteroidal anti-inflammatory drugs (NSAID) that has been developed for the treatment of osteoarthritis, rheumatoid arthritis, and pain (Riendeau et al. 2001). NSAIDs work by blocking the production of prostaglandins in response to injury or certain diseases (Michael et al. 2002) that cause pain, swelling, and inflammation. However, cyclooxygenase produces not only those prostaglandins that cause inflammation, but also certain prostaglandins that have useful roles in the body such as cytoprotection and therefore prevent mucosal ulceration and bleeding in the gastric intestinal tract. There are two different isoforms of cyclooxygenase enzymes, COX-1 and COX-2: COX-2 is the form that, among other things, produces prostaglandins causing inflammation, and COX-1 is the housekeeping enzyme, but does produce other prostanoids that have useful effects (Michael et al. 2002). Traditional NSAIDs, such as aspirin, ibuprofen, or diclofenac, block the action of both COX-1 and COX-2, and this is why they can sometimes cause side effects such as stomach irritation and peptic ulcers. Etoricoxib belongs to a new generation of NSAIDs that selectively blocks the action of COX-2 while sparing the action of COX-1. COX-2 can be expressed in human and animal colon cancer cells as well as in human colorectal adenocarcinomas (Tsujii et al. 1997). Also, the COX-2 enzyme plays a crucial role in the maintenance of gastrointestinal mucosal integrity, particularly when the mucosa is ulcerated or inflamed, which may lead to cancer (Wallace 1999). In recent times, studies have shown that conventional NSAIDs as well as selective COX-2 inhibitors inhibit chemically induced carcinogenesis in rats and mice (Kawamori et al. 1998a). However, the highest tolerated dose of nonselective NSAIDs typically reduced the number and size 53


of tumors by only 40% to 60%. Further, nonselective NSAIDs suppressed but did not completely eliminate the growth of chemically induced adenomatous polyps and cancers. In comparison, however, two studies with COX-2–specific NSAIDs called the coxibs have been found to inhibit 90% of tumors in rats and are better tolerated than comparable doses of nonselective NSAIDs (Kawamori et al. 1998b). Keeping this background in mind, the present study was designed to examine the chemopreventive efficacy of a COX2–specific inhibitor, etoricoxib, in rat intestine during 1,2dimethylhydrazine (DMH) supplementation on the biochemical estimations, antioxidative defense status, and membrane lipid composition, along with membrane dynamics studies. Although the morphogenesis of chemically induced neoplasm had been described both in the colon as well as the small intestine (Savov and Donchev 1991; Kobaeck-Larson et al. 2004), the results suggest for certain indications the molecular structure of the membrane in the small intestine as a cause to the refractoriness of this segment to the development of neoplastic growth.

MATERIALS AND METHODS Animals and Treatment Male Sprague Dawley rats (170–210 g) were obtained from the Central Animal House of Panjab University, Chandigarh. All the animals were kept in polypropylene cages under hygienic conditions and supplied with pellet diet and drinking water ad libitum. The rats were randomized into three groups. Rats in Group 1 served as control, which received the vehicle of the drugs, 1 mM EDTA-saline. Rats in Group 2 were administered freshly prepared DMH (Sigma Chemical Co., St. Louis, MO), 30 mg in 1 mM EDTA-saline, pH 6.5/kg body weight/week, subcutaneously. Group 3 animals received DMH + a daily oral dose of etoricoxib, 6 mg/kg body weight. The etoricoxib was received as pure salt through the courtesy of Ranbaxy Pharmaceuticals Co. (Gurgaon, India). The treatment was terminated at the end of 6 weeks and all the animals were sacrificed by anesthetizing with ether after an overnight fasting. All of the animal procedures as reported here followed the guidelines approved by the Panjab University Ethical Committee on the use of experimental animals for biomedical research.

Preparation of Intestinal Brush Border Membrane (BBM) The BBM of rat intestine was isolated using the method of Schmitz et al. (1973). A known weight of jejunum portion of the intestine was flushed with ice-cold saline, minced, and then homogenized in chilled 1 mM Tris–50 mM mannitol buffer (pH 7.4) in a motor-driven homogenizer at 4◦ C. The 10% homogenate was passed through two layers of cheese cloth. To the above filtrate, anhydrous CaCl2 was added with constant stirring (10 mM final conc.) on a magnetic stirrer and left for 10 to 15 min in cold. Later it was centrifuged at 2000 × g for 10 min at 4◦ C. The pellet thus obtained was discarded and the supernatant was recentrifuged at 42000 × g for 20 min. The supernatant obtained in the above step was discarded, while the pellet was suspended in 20 vol. of 50 mM sodium N. Mittal et al.

maleate buffer (pH 6.5–6.8) and recentrifuged at 42000 × g for 20 min. The supernatant was again discarded and the pellet was suspended in 50 mM sodium maleate buffer (pH 6.5–6.8) containing 0.02% sodium azide (NaN3 ). The final membrane obtained was similar to the P2 fraction of Schmitz et al. and used for various biochemical studies.

Assay of Disaccharidases The activity of sucrase, lactase, and maltase was determined by measuring the D- glucose liberated from the respective disaccharide sugar substrate using a glucose oxidase-peroxidase enzymatic system (GOD-POD) by Dahlqvist (1964).

Assay of Alkaline Phosphatase Alkaline phosphatase activity was assayed according to the method of Bergemeyer (1963) by measuring the liberated inorganic phosphate from the phosphate monoester substrate, p-nitrophenyl phosphate.

Lipid Peroxide (LPO) Lipid peroxide formation was assayed by the method of Wills (1966). Since malonyldialdehyde is a degradation product of peroxidized lipids, the development of pink color with the absorption maximum at 532 nm as a TBA-MDA chromophore has been taken as an index of lipid peroxidation.

Reduced Glutathione (GSH) Glutathione content was estimated according to the method of Ellman (1959). In this method 5,5-dithiobis-2-nitrobenzoic acid (DTNB) is reduced by –SH groups to form 1 mol of 2nitro-5-mercapto benzoic acid per mole of SH, which can be measured at 412 nm. The molar extinction coefficient for GSH at 412 nm is 13.6M−1 cm−1 , which was used for the calculation, and the results expressed as nmoles of GSH per mg protein.

Total Glutathione This analysis was done by the method of Zahler and Cleland (1968). The method is based on the reduction with dithioerythritol and determination of resulting monothiols with DTNB in the presence of arsenite. The arsenite forms a light complex with dithiols and not with monothiols. The absorbance was recorded at 412 nm and calculated as above.

Oxidized Glutathione (GSSG) Oxidized glutathione was quantitated by subtracting the values of reduced glutathione from the total glutathione levels.

Redox Ratio (GSH/GSSG) The redox ratio for all the groups was calculated by taking the ratios of the respective values of reduced glutathione to oxidized glutathione, as above. 54


Superoxide Dismutase (SOD)

Protein Estimation

Superoxide dismutase assay was performed according to the method of Kono (1978). The reduction of nitro blue tetrazolium (NBT) to a blue color formation mediated by hydroxylamine hydrochloride was measured under aerobic conditions. Addition of SOD inhibits the reduction of NBT and a 50% reduction is taken as a measure of the enzyme activity.

Protein concentration was determined by the method of Lees and Paxman (1972), using bovine serum albumin (BSA) as standard.

Catalase Catalase was estimated in a UV spectrophotometer by the method described by Luck (1971) using H2 O2 as a substrate. The absorption of H2 O2 solution is measured at 240 nm on decomposition of H2 O2 with catalase, and a decrease in absorption recorded.

Glutathione Reductase (GR) The enzyme was assayed by the method of Massey and Williams (1965). The utilization of NADPH at 340 nm is directly related to the activity of GR.

Glutathione S-Transferase (GST) The enzyme was assayed by the method of Habig et al. (1974). GST catalyzes the formation of the glutathione conjugates of CDNB, which absorb maximum at 340 nm and have an extinction coefficient of 9.6 mM−1 cm−1 .

Nitric Oxide (NO) NO production was estimated by the method described by Stuehr and Marletta (1987) by measuring nitrite, a stable metabolic product of NO, using the Griess reagent. NO synthase converts L-arginine to L-citrulline and NO, which quickly reacts with oxygen to yield nitrite, and then reacts with Griess reagent to form a purple azo dye the color of which can be read at 540 nm.

Citrulline Citrulline was estimated by the method of Boyde and Rahmatullah (1980). The citrulline assay was based on its reaction with diacetylmonoxime and absorbance measured at 530 nm.

Nucleic Acids (DNA/RNA) Nucleic acids (DNA and RNA) were assayed after extracting with perchloric acid and the absorbance was measured at 260 nm in a UV spectrophotometer by the method of Munro and Fleck (1966). The amount of nucleic acids in the sample was calculated using the following relationship: 0.1 O.D.unit = 128 µg RNA and 4.5 µg DNA/mL of the sample in the original solution 55

Extraction of Lipids Lipids were extracted from the BBM following the method of Folch et al. (1957). Membrane suspension (150–200 mg protein) was mixed in a flask with 20 vol. of chloroform:methanol (2:1 v/v) and left for 15 min at 45◦ C. The contents were mixed thoroughly and filtered through a Whatman No. 1 filter paper into a graduated cylinder. The residue left on the filter paper was then washed three times with 10 mL of chloroform:methanol (2:1). Then, 0.2 vol. KCl (0.9%) was added (20% of total volume) to the extract. The contents were mixed vigorously and allowed to stand in cold overnight so as to separate the aqueous and lipid layers distinct. Upper aqueous phase was removed with a Pasteur pipette and the lower layer washed three times with 2 mL chloroform:methanol:0.9% KCl, 3:48:47 v/v. The washed lower layer was transferred to a round bottom flask and evaporated to dryness at a temperature below 45◦ C, while the upper aqueous layer was added each time to the previously separated upper phase and used for the estimation of ganglioside sialic acid. To the residue, 5 mL of chloroform:methanol:water, 64:32:4 v/v, was added and evaporated to dryness. This was repeated three times. The dried lipid was redissolved in chloroform and filtered again. The filtrate was evaporated in a rotary evaporator under reduced pressure and at a temperature slightly