Abstract
The present study was designed to evaluate the effects of three nonsteroidal anti-inflammatory drugs (NSAIDs) with varying cycloxygenase selectivities on the small intestinal antioxidant enzyme status and surface characteristics during 1,2-dimethylhydrazine (DMH) administration. Male Sprague-Dawley rats were divided into five different groups: Group 1 (control, vehicle treated); group 2 (DMH treated, 30 mg/kg body weight/week, subcutaneously); group 3 (DMH + aspirin 60 mg/kg body weight); group 4 (DMH + celecoxib 6 mg/kg body weight); group 5 (DMH + etoricoxib 0.64 mg/kg body weight). Postmitochondrial fraction were isolated from the intestinal segments and different oxidative parameters and other parameters studied, such as the lipid peroxides, reduced and total glutathione, superoxide dismutase, catalase, glutathione reductase, glutathione S-transferase, nitric oxide, citrulline, and nucleic acids. At the end of 6 weeks of treatment, the results indicated a significant alteration in the antioxidative defense status of the intestine in the presence of the procarcinogen DMH, which was restored with the administration of NSAIDs. The study, therefore, suggests a possible mechanism for the chemopreventive effects of NSAIDs against the experimental intestinal cancer in rats.
Prostaglandins (PGs) have long been known to contribute to pain and inflammation in rheumatic diseases, as well as cytoprotection in the gastrointestinal tract. The contribution of prostaglandins in the inflammatory disorders is adequately controlled by nonsteroidal anti-inflammatory drugs (NSAIDs), by virtue of their inhibition of the PG synthesis. NSAIDs are among the most commonly used drugs for the treatment of pain and inflammation in rheumatic diseases and other musculoskeletal disorders. Several reviews (Janne and Mayer 2000) have summarized the accumulating evidence that NSAIDs have great promise as anticancer drugs. The chemopreventive efficacy of NSAIDs against colorectal cancer has particularly been well studied (Giardiello, Offerhaus, and DuBois 1995). Also, NSAIDs may decrease the incidence of carcinomas of the esophagus, stomach, breast, lung, prostate, urinary bladder, and ovary (Thun, Henley, and Patrono 2002). NSAIDs have been shown experimentally to stimulate apoptosis and to inhibit angiogenesis, two mechanisms that help to suppress malignant transformation and tumor growth. However, to date the clinical use of these agents is limited only to patients with familial adenomatous polyposis (FAP), which may benefit from the chemopreventive treatment with the selective cycloxygenase (COX) inhibitors (Marx 2001).
However, NSAIDs are also associated with tissue toxicities such as gastrointestinal ulcers, bleeding, and sometimes gastric perforation due to deep ulceration. The ulcerogenic properties of NSAIDs are not solely explained by the inhibition of COX-1 and require the inhibition of both COX-1 and COX-2, suggesting a role for COX-2 as well as COX-1 in maintaining the integrity of the gastrointestinal mucosa (Tanaka et al. 2001, 2002). These toxicities hampered the long-term use of classical NSAIDs for chemoprevention and thereby remove the cytoprotective function of the prostaglandins in the intestinal mucosa. Toxicity may be due to initial biochemical modifications or due to alterations in the intestinal antioxidative defense mechanism. Also, gastrointestinal toxicities due to aspirin have been suggested by various reports (Roderich, Wilkes, and Meade 1993). Thus, selective COX-2 inhibitors (celecoxib and etoricoxib) may become more effective and safer chemopreventive agents, which spare the COX-1, and thereby the intestinal toxicity is prevented. However, before being accepted for clinical use, these drugs need to be further evaluated for membrane damage and related surface changes.
MATERIALS AND METHODS
Animals and Treatment
Male Sprague-Dawley rats (170 to 210 g) were obtained from the central animal house of the Panjab University, Chandigarh. All the animals were kept in polypropylene cages under hygienic conditions and supplied with pellet diet and drinking water ad libitium. The animals were divided into five groups, each containing eight rats: Group 1 (control) received the vehicle of the drugs, 1 mM EDTA–saline. Group 2 (DMH treated) was administered freshly prepared DMH (1,2-dimethylhydrazine; Sigma Chemical, St. Louis, MO, USA), 30 mg/kg body weight/week, subcutaneously. Group 3 was given DMH + a daily oral dose of aspirin at 60 mg/kg body weight. Group 4 was given DMH + a daily oral dose of celecoxib at 6 mg/kg body weight. Group 5 was given DMH + a daily oral dose of etoricoxib at 0.64 mg/kg body weight. The NSAIDs, aspirin, celecoxib, and etoricoxib were received as pure salt through the courtesy of Ranbaxy Pharmaceuticals, Gurgaon, India, and the doses of the NSAIDs were chosen within their therapeutic anti-inflammatory range as based on reported ED50values for rats (Kanwar et al. 2007). After 6 weeks of treatment, the animals were anesthetized with ether and sacrificed quickly by decapitation. All of the animal procedures as reported here followed the guidelines approved by the Panjab University Ethical Committee on the use of the experimental animals for biomedical research. The principles of animal care as laid down by the National Institute of Health (NIH publication no. 23–85, revised in 1985) have been broadly followed.
Tissue Homogenization
A 10% homogenate of the small intestinal segment was prepared in chilled 1 mM Tris–50 mM mannitol buffer (pH 7.4).The homogenate was centrifuged at 1000 ×g for 10 min at 4°C. Pellet was discarded and the supernatant used for lipid peroxidation and reduced glutathione estimations. A portion of 1000 × g supernatant was again centrifuged at 10,000 × g for 20 min to obtain the post mitochondrial supernatant (PMS), which was further used for various biochemical estimations and antioxidative enzyme assays.
Lipid Peroxide (LPO)
Lipid peroxide formation was assayed by the method of Wills (1966). Because malonyldialdehyde (MDA) is a degradation product of peroxidized lipids, the development of pink colour with the absorption characteristics (absorption maximum at 532 nm) as a 2-thiobarbituric acid-MDA (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-nitro)benzoic acid (DTNB) is reduced by –SH groups to form 1 mole of 2-nitro-5-mercaptobenzoic acid per mole of SH, which can be measured at 412 nm. The molar extinction coefficient for GSH at 412 nm is 13.6 M−1cm−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 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)
Redox ratio for all the groups was calculated by taking the ratios of the respective values of reduced glutathione to oxidized glutathione, as above.
Superoxide Dismutase (SOD)
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.
Catalase
Catalase was estimated in an ultraviolet (UV) spectrophotometer by the method described by Luck (1971) using H2O2 as a substrate. The absorption of H2O2 solution is measured at 240 nm on decomposition of H2O2 with catalase, and a decrease in absorption is 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, Pabst, and Jakoby (1974). GST catalyzes the formation of the glutathione conjugates of 1-chloro-2,4-dinitrobenzene (CDNB), with absorption maximum at 340 nm and have an extinction coefficient of 9.6 mM−1cm−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
Citrulline
Citrulline was estimated by the method of Boyde and Rahmatuleen (1980). The citrulline assay based on its reaction with diacetylmonooxime and absorbance was measured at 530 nm.
Nucleic Acids (DNA/RNA)
Nucleic acids (DNA and RNA) were assayed with perchloric acid and absorbance was measured at 260 nm in an UV spectrophotometer by the method of Munro and Fleck (1966). The amount of nucleic acid in the sample was calculated using the following relationship:
Protein Estimation
Protein concentration was determined by the method of Lees and Paxman (1972) by using bovine serum albumin (BSA) as standard, which is an adoption of the Lowry method.
Statistical Analysis
Statistical analysis of the data was performed by one-way analysis of variance (ANOVA) following post hoc test using least significance data (LSD), using SPSS software package.
RESULTS
Lipid Peroxidation and Antioxidant Enzymes
Shown in Table 1, no significant alteration was observed in DMH treatment when compared with the control group. Similarly, no significant changes were noted in all the treatment groups when compared with the DMH-treated group.
DMH treatment per se did not produce any change in SOD activity when compared to the control group. In comparison to DMH treatment, a highly significant increase (p < .001) in the activity of SOD was observed in both the treatment groups, i.e., aspirin and etoricoxib, whereas a nonsignificant rise was seen in celecoxib treatment.
A highly significant decrease (p < .001) was shown by DMH treatment group in the catalase activity when compared to control. Aspirin-treated group showed a highly significant (p < .001) rise in the catalase activity when compared to the DMH. A fairly significant increase (p < .05) was shown in the catalase activity by celecoxib, whereas etoricoxib showed a significant (p < .01) increase in comparison to DMH-treated group.
The glutathione reductase activity was significantly (p < .01) increased in DMH treatment when compared to control. When compared with DMH treatment, only the celecoxib-treated group showed a significant (p < .01) decrease in the glutathione reductase activity, whereas none of the other treated groups showed any significant change.
A highly significant decrease (p < .001) was observed in the levels of GST in DMH-treated group when compared with the control. In comparison to DMH treatment, aspirin showed a highly significant increase (p < .001), whereas no significant change was observed in celecoxib treatment. A significant increase (p < .01) was, however, seen in etoricoxib treatment.
Nonenzymatic Antioxidant Defense System
DMH treatment showed a significant decrease (p < .01) in the GSH levels when compared with the control group (Table 2). In comparison to DMH treatment, aspirin-treated group showed a significant increase (p < .01) in the GSH level, whereas both the celecoxib- and etoricoxib-treated groups resulted in a highly significant increase in the GSH levels (p < .001).
No significant change in the level of total glutathione was seen in DMH treatment when compared with the control group. When compared to DMH-treated group, a highly significant increase (p < .001) was shown by both aspirin- and etoricoxib-treated groups, whereas no significant change was shown by celecoxib treatment.
DMH treatment brought about a significant increase (p < .01) in the level of oxidized glutathione when compared with the control group. In comparison to DMH treatment, a highly significant increase (p < .001) was shown by aspirin-treated group, whereas a decrease was observed in the oxidized glutathione levels up to the same level of significance (p < .001) in the celecoxib-treated group. Etoricoxib showed a highly significant (p < .001) decrease in the levels of oxidized glutathione.
No significant change was observed in any of the treatment group when compared with the control and with the DMH treatment groups, except the etoricoxib treatment group. A fairly significant increase (p < .05) was observed in the GSH/GSSG ratio in etoricoxib when compared with the DMH-treated group.
Nitric Oxide Synthase Activity and Nucleic Acid Level
Nonsignificant alteration was found in the NO synthase activity in the DMH treatment group when compared with the control group, as shown in Table 3. When comparisons were made with the DMH-treated group only, the celecoxib-treated group showed a fairly significant increase (p < .05) in the NO synthase activity.
In comparison to DMH-treated group, both celecoxib and etoricoxib showed significant changes. The celecoxib-treated group registered a significant increase (p < .01) in the citrulline level, whereas the etoricoxib showed highly significant (p < .001) increase. The aspirin-treated group showed a non-significant rise in the citrulline level.
Table 3 also shows that no significant change was observed in nucleic acid content in any of the treatment groups, when compared with the control group as well as the DMH-treated group.
DISCUSSION
The present study indicated the alterations in antioxidant defense status in a procarcinogen DMH treatment reflecting the oxidative stress in the intestinal epithelium. Lipid peroxidation has been implicated in the pathogenesis of a variety of diseases including cancer (Ray and Hussian 2002). In the present study, however, no significant change had been observed in the different NSAID treatments with DMH administration, except in etoricoxib treatment. This shows that there is not much increase in the MDA level, resulting in lesser production of reactive oxygen species, which is responsible for the elevated lipid peroxidation and also restoring the activities of enzymatic antioxidants.
The level of reduced glutathione (GSH) is considered a critical determinant for the threshold of tissue injury caused by environmental chemicals (Meister and Tate 1976). The depletion in GSH levels may occur due to the induction of oxidative stress by DMH treatment, possibly by interfering with the glutathione redox cycle. The enhancement in the GSH level during the treatment with NSAIDs speculates that they protect the cellular constituents from the attack of peroxides and free radicals. It has long been known that glutathione can be reversibly oxidized to gluthatione disulfide (GSSG). Also, glutathione peroxidase catalyzes the interaction of glutathione with hydrogen peroxide and other peroxides to yield glutathione disulfide. In the present study, oxidized glutathione was found to be increased in aspirin treatment along with DMH, which suggests the involvement of increased oxidative stress. Further, with celecoxib and etoricoxib treatments, the level of oxidized glutathione was found to decline, which may indicate the antioxidative property of the selective COX-2 NSAIDs. Similarly, the total glutathione level was found to be increased in all the treatments. Glutathione reductase plays an important role in maintaining the ratio of GSH/GSSG, which is considered crucial for the redox status of the cells. No significant change was observed in the redox ratio of GSH/GSSG except in the etoricoxib treatment, which shows a slight increase. It is difficult to correlate the GSSG level and this enzyme activity in the cells; however, it was noted that the activity of the enzyme was regulated both by NADPH and GSSG levels (Eggleston and Krebs 1974).
Superoxide radicals (O2 −) have been implicated in several pathological disorders and to be responsible for elevated oxidative stress (Fridrovich 1986). SOD and catalase are both antioxidant enzymes that function as blockers of free radical process (Dormandy 1978). Results of the present work have shown a slight decrease in the level of SOD in the DMH-treated group, indicating oxidative stress. It might result from the decreased synthesis of SOD, which is thus not able to scavenge the free radicals. Further, all the NSAID-treated groups have shown increase in SOD levels, which is due to the highly COX inhibitory nature and free radical–scavenging capabilities of celecoxib and etoricoxib, which protect the cellular environment from reactive oxygen species by increasing the enzyme synthesis. Numerous studies have shown the importance of SOD in protecting the cells against oxidative stress (Huang et al. 1997). A decline in the catalase activity was observed during DMH treatment. The inhibition of catalase activity is suggestive of enhanced synthesis of superoxide anion, which is a powerful inhibitor of catalase. It has been reported earlier that during tumorigenesis, catalase activity declines (Jaruga and Olinski 1994). The supplementation of NSAIDs showed considerable improvement in catalase activity, which may be attributed to their indirect effects in replenishing the antioxidant enzyme activities.
The GSH-related enzymes glutathione reductase (GR) and glutathione S-transferase (GST) are known to play a major role in free radical scavenging. GR is involved indirectly in the protection of cells from the adverse effects of oxidative damage (Kaneko et al. 2002). In the present study, the increase in enzyme activity may be required to maintain the desired levels of GSH to tackle the conditions of stress, as it is known to utilize GSH as a substrate to catalyze the reduction of organic hydroperoxides and hydrogen peroxides (Ray and Hussian 2002). GSTs are also very important detoxification enzymes (Armstrong 1997) and protect the cells from the damaging effects of reactive oxygen species (ROS) (Hayes and Pulford 1995). In the present study, the decline in enzyme activity points toward the generation of ROS by DMH administration. The enzyme catalyzes the detoxification through the formation of GSH conjugates (Sies and Ketterer 1988). Therefore, reduction in GST activity may be due to the lesser availability of GSH, which is also found to be depleted in the present study. The enhancement in the activity of GSTs in all the NSAID treatment during DMH administration suggests the neutralization of ROS by NSAIDs.
The role of nitric oxide (NO) in carcinogenesis is controversial, as NO has both antitumor and tumor-promotive properties (Ricciardolo et al. 2004). It has been reported that very high levels of NO can be detrimental to the survival of certain tumor cells because of NO-mediated cellular injury, cytostatics, and apoptosis, whereas sustained release of low to moderate levels of NO may promote cellular invasiveness and differentiation (Pipili-Syntos et al. 1994). In the present study, 6-week treatment of DMH alone or in combination with different NSAIDs resulted in no change in the NO levels in the small intestine. There are two citrulline production pathways: it can be derived from arginine/NO synthase (NOS) pathway and/or arginine/arginase pathyway (Wu and Morris 1998). The NOS pathway is responsible for the production of NO as well as citrulline as the end product. Both NO and citrulline play major role in cell growth and proliferation. In the present study, citrulline levels were found to be unaltered with reference to control following various treatments except for the low dose of etoricoxib, which resulted in an enhancement of the citrulline levels. Further, when DMH along with NSAIDs were compared with the DMH treatment alone, both celecoxib and etoricoxib resulted in an enhanced citrulline production. The mere production of citrulline following NSAIDs administration and not of NO may be suggestive of more arginase-dependent pathway of citrulline formation.
Antioxidant protection can also operate by not repairing excessively damaged molecules to minimize the introduction of mutations. During normal oxygen metabolism, antioxidative enzymes eliminate toxic reduction intermediates of oxygen inside the cell, allowing a small pool of low-molecular-mass iron (Jacobs 1970) to safely exist for synthesizing DNA and iron-containing proteins, as well as signaling functions. In the present work, no variations were seen in nucleic acid content, suggesting that oxidative stress does not causes any damage to nucleic acid synthesis. Earlier studies have shown, however, that lipid peroxidation also resulted in DNA damage (Higuchi 2003).
Footnotes
Tables
Acknowledgements
The authors thank Panjab University for providing funds to the Department of Biophysics and also to the University Grant Commission–Special Assistance Programme.