The aim of this study was to investigate the effect of Chloroquine phosphate (at therapeutic dose) on existing gastric ulceration in albino rats. Rats were treated with Chloroquine phosphate (8.5 mg kg-1) intramuscularly for 24 h after formation of ulcers induced by acidified ethanol and indomethacin. Following sacrifice, colorimetric assays were applied to determine the concentration of protein and mucus, activities of catalase and lipid peroxidation in homogenized gastric mucosal samples. Chloroquine phosphate worsens gastric lesions produced by both indomethacin and acidified ethanol. Also, it seemed to elaborate the indomethacin and acidified ethanol induced effects on gastric juice volume, pH and acid output. On the other hand, thiobarbituric acid reactants (TBAR) was further increased and protein, catalase and mucus were decreased in the gastric mucosal samples. The data indicates that the use of Chloroquine may be dangerous to the integrity of the stomach, especially in existing gastric ulcers. It increases oxidative stress in the gastric mucosa caused by indomethacin and acidified ethanol.
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Chloroquine is a 4-aminoquinoline derivative used, particularly in the sub-Saharan Africa, as a cheap antimalarial and for the treatment of rheumatoid arthritis, lupus erythematosus and various inflammatory conditions (Onigbogi et al., 2000).
In spite of reports of increasing resistance to it by Plasmodium falciparum Chloroquine still remains the most prescribed antimalarial medication in most Sub-Saharan African countries (Olliaro and Taylor, 2003). This may be due to its affordability and availability across the tropics. Also, misuse is widespread (White, 2004). Although well tolerated in most individuals, Chloroquine is associated with pruritus (Ajayi et al., 1989; Onigbogi et al., 2000).
Hypotension induced by parenteral administration of Chloroquine is a common and serious adverse effect of this drug. It stimulates the release of histamine, as evidenced in the cause of venodilation (Abiose et al., 1997). Besides, it promotes attenuation of cyclic adenosine monophosphate (cAMP) and an increase in nitric oxide (NO) production in renal functions (Ahmed and Osman, 2007). Lysosomal dysfunction (Mahon et al., 2004), aberrations in serum proteins and free amino acids (El-Sayed et al., 1998) and various lipidemic effects (Gafaar et al., 1995) are all Chloroquine`s toxicological consequences on the retina.
Chloroquine stimulates gastric acid secretion (Etimita et al., 2005), which may not be beneficial in peptic ulceration.
Peptic ulceration results from increased formation of Reactive Oxygen Species (ROS) and/or decreased antioxidant reserve in the gastric mucosa, a condition termed as oxidative stress.
Hence, its occurrence is associated with oxidative stress increases by pro-ulcerative factors in the gut like Helicobacter pylori (Yamaguchi and Kakizoe, 2001), use of Non steroidal anti-inflammatory drugs, NSAIDs (Rostom et al., 2000), smoking (Ma et al., 2000), psychological stress (Mawdsley and Rampton, 2006), lead exposure (Olaleye et al., 2007) and dietary intake of potential ulcerogens (Ibironke et al., 1997).
From available literature, there seems to be a dearth of information on possible role of use and/or misuse of chloroquine in the etiology of gastric ulceration. We, therefore, present findings on the potentiation of gastric mucosa injury by therapeutic dose of chloroquine in albino rats.
MATERIALS AND METHODS
This study was carried out between July and August 2007 in the Department of Physiology, School of Basic Medical Sciences, Igbinedion University, Okada, Edo State, Nigeria.
Drugs: Chloroquine phosphate used were obtained from a local pharmacy duly registered by the Pharmacists` Council of Nigeria (PCN). Indomethacin was obtained from Strides, Belgium. All other reagents were of analytical grade and obtained from the British Drug Houses, Poole, UK.
Animals: Forty healthy adult albino rats of Wistar strain weighing between 180-220 g each were used in the study. The animals were housed under standard conditions of temperature (23±2°C), humidity (55±15%) and 12 h light (7.00 am-7.00 pm).
They were kept in wire meshed cages and fed with commercial rat pellets (Ladokun Feeds Ltd., Ibadan, Nigeria) and allowed water ad-libitum.
Treatment: The animals were divided into five groups with eight rats each. Group 1 was treated with normal saline after 36 h fasting. Group 2 was treated with indomethacin (40 mg kg-1) orally after 36 h fasting. Group 3 was treated with 1.0 mL HCl/Ethanol mixture (0.15 N HCl in 70% ethanol). Group 4 and 5 received Chloroquine intramuscularly (8.5 mg kg-1) 4 h after indomethacin and acidified ethanol administration.
Ulcer induction and index determination
Indomethacin induced ulceration: Indomethacin (Strides, Belgium) dissolved in sodium bicarbonate was administered orally (40 mg kg-1) to 36 h fasted rats.
Four hours later (for the treated control) and 24 h later (for the experimental group), the animals were killed with ether anaesthesia. The stomachs were opened along the greater curvature, washed in normal saline to remove debris and pinned on a cork mat for ulcer scoring. This was done by locating the wounds in the glandular regions under a simple microscope. The lengths (mm) of all the elongated black-red lines parallel to the long axis of the stomachs in the mucosa was measured. The ulcer index was calculated by adding the lengths of all the lesions in the glandular region of the stomach (Rifat-uz-Zaman et al., 2004; Tanaka et al., 1993). The wounds were assessed independently by three observers.
HCl/Ethanol induced ulceration: Thirty-six hours fasted rats were given 1.0 mL HCl/Ethanol mixture containing 0.15 N HCl in 70% ethanol (Anadan et al., 1999). Four hours later (for the treated control) and 24 h later (for the experimental group), the animals were sacrificed under sodium pentobarbitone anaesthesia (60 mg kg-1 i.p).The stomachs were removed ,inflated with 10 mL of 2% formaldehyde for 10 min to fix the tissue walls and opened along the greater curvature. The hemorrhagic lesions were stretched out on a glass plate and their sizes were estimated using an underlying graph study with a 1 mm2 grid. Lesions areas were summed up per stomach and expressed as % of the total mucosal area.
Gastric juice volume, pH and acid output: The four hour gastric juice collection was drained into a graduated test tube and centrifuged at 2000 rpm for 10 min. The supernatant volume and pH were recorded. The total acid content of the gastric juice was also determined by titration to pH 7.0 with 0.05 N NaOH, using phenolphthalein as indicator.
The protein content was also estimated as described by Lowry et al. (1951).
Determination of lipid peroxidation: Lipid peroxidation was assayed by measuring the thiobarbituric acid reactants (TBAR) products using the procedure of Walls et al. (1976). Briefly, the homogenate was supplemented with 0.75 g L-1 TBA in 0.1 mol L-1 HCl. The reactants were then supplemented with 5 mL n-butanol-pyridine mixture, shaken vigorously for 1 min and centrifuged for 10 min at 4000 rpm. Absorbance was then read at 532 nm and the results expressed as nmol TBA/100 mg wet tissue.
Determination of catalase activity: Activity of catalase in gastric mucosa was determined according to the procedure of Sinha (1972). This method is based on the reduction of dichromate in acetic acid to chromic acetate when heated in the presence of H2O2, with the formation of perchromic acid as an unstable intermediate. The chromic acetate so produced is measured calorimetrically at 530 nm.
Determination of gastric mucous: Adherent gastric glandular mucous was measured by the method of Corne et al. (1974). The excised stomachs were soaked for 2 h in 0.1% Alcian blue dissolved in buffer solution containing 0.1 M sucrose and 0.05 M Sodium acetate (pH adjusted to 5.8 with hydrochloric acid). After washing the stomach twice in 0.25 M sucrose (15 and 45 min), the dye complexed with mucous was eluted by immersion in 10 mL aliquots of 0.5 M MgCl2 for 2 h. The resulting blue solution was shaken with equal volumes of diethyl ether and the optical density of the aqueous phase measured at 605 nM using a spectrophotometer.
Using a standard curve, the absorbance of each solution was then used to calculate the various concentration of the dye and the weight of dye (expressed in mg). The weight of the dye was then expressed over the weight of the stomach.
Statistical analysis: The data obtained were expressed as Means±SEM (Standard Error of Means of eight experiments) and analysed statistically by application of the Statistical Package for Social Sciences (SPSS).
The student`s t-test was applied and p-values were determined. Differences were considered significant at p<0.05 and highly significant at p<0.001.
RESULTS AND DISCUSSION
Indomethacin ,and acidified ethanol causes increased gastric secretion volume and acid output, with significant decreased gastric pH (Table 2, 4). Similarly, gastric ulcer lesions were formed by indomethacin and acidified ethanol in the experimental rats (Table 1, 3) (p<0.001). This is in agreement with the report of several authors on the role of indomethacin and acidified ethanol on gastric ulcer and erosion formation (Christopher et al., 1998). Also, gastric mucous and protein were significantly decreased in the indomethacin (Table 1) and acidified ethanol (Table 2) treated gastric mucosa, which is not contradictory to the findings of Rao et al. (1997) and Desai et al. (1997).
Chloroquine administration on existing ulcer in the rat led to an increase in the ulcer index; 13.75±1.59 mm (when compared with 8.58±1.44 mm index of the indomethacin treated group) as shown in Table 1 (p<0.001) and 10.20±0.45% (when compared with 7.15±0.55% mucosa damage in the acidified ethanol treated group) as shown in Table 3 (p<0.05), with further decrease in barrier mucus and protein content of the gastric mucosa (Table 1, 3), p<0.05.
In addition, Chloroquine seemed to elaborate the gastric changes induced by indomethacin and acidified ethanol. This is depicted in Table 2 and 4.
Figure 1 and 2 show the lipid peroxidation and catalase activity in the ulcerated gastric mucosa after chloroquine treatment, expressed as percentage change of the control value. Lipid peroxidation is measured as the amount of TBA reactants in the gastric mucosa. In the indomethacin treated control animals, it was 620±71 mmol g-1 tissue. Chloroquine significantly increased gastric TBA reactants level to 51.2±2.3% of the control value. Similarly, in the acidified ethanol treated control animals lipid peroxidation was 640±37 mmol g-1 tissue, which was significantly increased on chloroquine administration to 110±10% of the control value.
|Table 1:||Effects of chloroquine treatment on indomethacin-induced gastric mucosa injury in the rat|
|Indomethacin: Highly significant from normal saline treated p<0.001**, significant p<0.05, Chloroquine: Highly significant from indomethacin treated p<0.001**, significant p<0.05*|
|Table 2:||Gastric juice profile of saline, indomethacin and chloroquine treated rats|
|Indomethacin: Highly significant from normal saline treated p<0.001**, significant p<0.05*, Chloroquine: Highly significant from indomethacin treated p<0.001**, significant p<0.05*|
|Table 3:||Effects of chloroquine treatment on acidified ethanol gastric mucosa injury in the rat|
|Acidified ethanol: Highly significant from normal saline treated p<0.001**, significant p<0.05*, Chloroquine: Highly significant from acidified ethanol treated p<0.001**, significant p<0.05*.|
|Table 4:||Gastric juice profile of saline, acidified ethanol and chloroquine treated rats|
|Acidified ethanol: Highly significant from normal saline treated p<0.001**, Chloroquine: Highly significant from acidified ethanol treated p<0.001**, significant p<0.05*|
|Fig. 1:||TBA reactants and catalase activities in ulcerated (indomethacin) gastric mucosa treated with chloroquine. (Mean±SEM, n = 8) (p<0.05)|
|Fig. 2:||TBA reactants and catalase activities in ulcerated (acidified ethanol) gastric mucosa treated with Chloroquine (Mean±SEM, n = 8) (p<0.05)|
On the other hand, chloroquine administration further decreases gastric mucosal catalase activity to 49.5±5.1% of indomethacin-treated control value and 60.5±2.5% of acidified ethanol treated control value.
The results of this study show a kind of relationship between the use of antimalarials and gastric ulceration.
Chloroquine phosphate, the first line defense synthetic drug in Nigeria and many African countries because of its commonality and cheap status, (Gustafsson et al., 1983), is observed to aggravate the existing ulcer in the stomach of the rat. Previous studies have shown that it caused significant increase in gastric acid secretion (Etimita et al., 2005), one of the risk factors in the development of ulcer. This is also, confirmed by the increase in acid output observed in the present study. It is well known that gastric acid secretion plays a role in gastric ulcer; which explains the mechanism of action of many anti-ulcer drugs (Schmassmann, 1998).
Lipid peroxidation, a result of the reaction of oxyradicals and polyunsaturated fatty acids, has been implicated in the etiology of damage to subcellular membranes and then injury in the cell.
In the present study, lipid peroxidation, as measured by the amount of TBA reactants, was increased on chloroquine administration. This implies that chloroquine causes an increase in the formation of free radicals, which, if not mopped up by free radical scavengers such as superoxide dismutase, catalase, or glutathione, will expose the stomach to inflammation.
Moreover, indomethacin and acidified ethanol has been known to cause lipid peroxidation (Kapui et al., 1993; Anadan et al., 1999), with depletion of endogenous antioxidants. The result of this study also showed a further significant decrease in barrier mucous and glandular protein level in the ulcerated gastric mucosa treated with chloroquine. This may not be unconnected with the low level of CAT observed in the study.
Gastrointestinal wall integrity is known to be controlled by two opposing forces: Defensive forces and the aggressive force (Corne et al., 1974). The defensive force are gastro protective and involve antioxidative enzymes; SOD which catalyses the dismutation of superoxide radical anion (O2-) into less noxious hydrogen peroxide (H2O2) and CAT or glutathione peroxidase that inactivate H2O2 by the degradation into water (Masuda et al., 1995). Depletion of CAT, as evidenced in this study underscore the predisposition of stomach to a greater impact of free radicals produced through increased lipid peroxidation. However, further studies are still needed to investigate the role of other anti-oxidative enzymes in the aggravation of existing ulcers by Chloroquine.
In conclusion, the results of the present study show that there is potentiation of ulceration in the stomach of rats treated with Chloroquine phosphate. If these findings are extrapolated to man, they suggest that indiscriminate and unguided use of Chloroquine by peptic ulcer-prone individuals, may be dangerous.
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