Effects of Vitamin E,Vitamin C,and Selenium on Gastric Fundus in Cadmium Toxicity in Male Rats

Abstract

Cadmium (Cd) is a highly toxic metal. It has an indirect role in the generation of various free radicals. Antioxidants such as vitamin E, vitamin C, and selenium are important for preventing the damage caused by reactive oxygen species. This study was undertaken to examine the effect of acute cadmium and/or antioxidants on serum lipid metabolism, tissue glutathione, and lipid peroxidation (LPO) levels, and ghrelin and metallothionein production in the gastric fundus mucosa of rats. Cd (2 mg/kg/day CdCl₂) was administered to rats for 8 days, intraperitoneally. Vitamin E (250 mg/kg/day) + vitamin C (250 mg/kg/day) + sodium selenate (0.25 mg/kg/day) were administered to rats orally at the same time. The animals were treated by antioxidants 1 h prior to treatment with Cd every day. Gastric tissue homogenates were used for protein and glutathione and LPO levels. Phospholipid and total lipid levels were determined in serum. Gastric fundus sections examined for histopathological changes and by immunohistochemistry for expression of ghrelin and metallothionein. In the group treated with Cd, degenerative changes such as discontinuity in the surface epithelium were observed. The degenerative changes induced by Cd were decreased in the group given vitamin E + vitamin C + selenium. There was no significant change in ghrelin- and metallothionein-immunoreactive cells in fundus mucosa. Stomach glutathione levels insignificantly decreased in the Cd groups, but in the Cd group given antioxidant, stomach glutathione levels were significantly increased. Serum phospholipid and total lipid levels were significantly increased in the Cd groups. On the other hand, treatment with antioxidants reversed these effects. These results indicate that antioxidants partly prevent the toxicity of Cd in rat gastric fundus.

Cadmium (Cd) is a well-known industrial and environmental toxicant (Volka et al. 2006), although less is known about the acute effects of cadmium on stomach. It produces oxidative stress by disturbing the prooxidant-antioxidant balance (Shaikh, Vu, and Zaman 1999). Substantial evidence suggested that in the liver, kidneys, testicles, and brain, cadmium toxicity is associated with a decrease in several antioxidants (Ercal, Gurer-Orhan, and Aykin-Burns 2001; Grosicki 2004; Volka, Morris, and Cronin 2005). High reactive oxygen species (ROS) concentrations can be the cause of altered gene expression and damage to cell structures, including lipids and proteins (Stohs, Bagchi, and Hassoun 2001; Poli et al. 2004). Nonenzymatic antioxidants as vitamin C, vitamin E, and glutathione (GSH) are the best protectors of the damage caused by ROS (Seifried et al. 2007). GSH is a multifunctional intracellular antioxidant and can alter cadmium excretion (Tandon, Singh, and Dhawan 1992; Masella et al. 2005). Selenium may inhibit oxidative stress–induced DNA damage (Battin, Perron, and Brumaghim 2006). Vitamin E, vitamin C, and selenium might modulate cadmium-induced toxicity through antioxidative mechanism. Ghrelin is the endogenous ligand of the G protein–coupled growth hormone secretagogue receptor. Ghrelin was originally isolated from the stomach. The regulation of the ghrelin secretion is largely unknown (Brzozowski et al. 2005). Metallothionein is a protein capable of binding metals and can scavenge ROS (Sato and Bremner 1993; Florianczyk 1996; Park, Liu, and Klaassen 2001). The aim of this study was to determine the changes in ghrelin and metallothionein peptides, lipid metabolism, and glutathione levels following exposure to cadmium with or without antioxidant supplementation for 8 days.

MATERIAL AND METHODS

Animals and Tissue Preparation

Adult 6.5– to 7-month-old male Sprague-Dawley rats, weighing 250 to 300 g (n = 59) were used. The experiments were reviewed and approved by the Local Institute’s Animal Care and Use Committees. All rats were fed with standart food pellets and tap water ad libitum, and were divided into four groups. Group I: intact control animals; group II: control rats administered vitamin E (α-tocopherol; Sigma) 250 mg/kg/day in 1 ml sunflower oil per animal + vitamin C (ascorbic acid, Sigma) 250 mg/kg/day in 0.5 ml distilled water per animal + sodium selenate (Se; Fluka) 0.25 mg/kg/day in 0.5 ml distilled water per animal; group III: animals administered only Cd (CdCl₂; Merck) 2 mg/kg/day in 1 ml distilled water per animal for 8 days; and group IV: rats administered vitamin E + vitamin C + selenium + Cd (in the same dose and at the same time). The antioxidants were administered to rats orally and cadmium was administered intraperitoneally. The animals were treated with antioxidants one hour prior to treatment with Cd every day.

Histopathology

On the 9th day of the experiment, all of the animals were fasted overnight and then sacrificed under ether anesthesia, and the fundus region of their stomachs was removed. The tissue samples were fixed in Bouin’s fixative, dehydrated in a graded series of ethanol, and embedded in paraffin wax before sectioning. The sections were stained with periodic acid–Schiff (PAS) reaction to visualize mucus-secreting cells in the fundus. In addition, the sections were stained with Masson’s trichrome dye and examined by light microscopy.

Immunohistochemistry

The paraffin sections were dewaxed and rehydrated. The tissues were rendered permeable with 0.3% Triton-X 100 for 10 min and then rinsed in phosphate-buffered saline (10 mM, pH 7.5). For antigen retrieval, the slides were kept in 0.01 M citrate buffer for 10 min in microwave oven. Endogenous peroxidase was blocked with 3% hydrogen peroxide. Ultra Vision Detection System for streptavidin-biotin-peroxidase technique (Lab Vision, USA) was employed. Sections were covered with blocking serum for 10 min to block nonspecific binding sites. They were then incubated with metallothionein antibody for 1 h (Zymed Laboratories, USA) at room temperature with 1:50 dilution. Slides were incubated with ghrelin goat polyclonal antibody for 1 h (Santa Cruz Biotechnology, USA) at room temperature with 1:50 dilution. ABC staining system (Santa Cruz Biotechnology, USA) was used. They were incubated with biotinylated secondary antibody for 15 min, and the avidin and biotinylated horseradish peroxidase for 15 min, respectively. The enzyme activity was developed using aminoethylcarbazole (AEC) and then the sections were counterstained with hematoxylin. Negative control sections were prepared by substituting the metallothionein or ghrelin antibodies with phosphate-buffer saline. The mean count of ghrelin-immunoreactive cells per field of vision by a light microscope was determined for each animal by averaging the number of cells in 10 randomly selected fields of vision in which the entire thickness of the mucosa was visible in a single section that was immunohistochemically stained. The significance of changes in ghrelin-immunoreactive cells was evaluated statistically using one-way analysis of variance (ANOVA). The number of ghrelin-immunoreactive cells was statistically not different (p = 0.29). As more metallothionein-immunoreactive cells were localized all through the mucosa we did not evaluate those statistically.

Biochemical Assays

Biochemical investigations were made in serum and tissue. The serum was separated from the blood and was kept at (−35°C). Serum total lipid levels were determined by the method sulphophosphovanilin (Frings et al. 1972). The serum was heated with concentrated sulfuric acid without prior deproteinisation, and was then mixed with phosphoric acid/vanillin reagent. In this sulfo-phospho-vanillin reaction, the serum lipids formed a pink dyestuff, which was measured spectrophotometrically measured at 530 nm. Serum phospholipid levels were determined by Zilversmit methods (Zilversmit and Davis 1950). Serum phosphatides were precipitated, together with the serum proteins, with trichloroacetic acid. The organic components of the precipitate were destroyed with perchloric acid with a small addition of nitric acid. The liberated phosphate reacted with molybdate, resulting in the formation of phosphomolybdate. This was reduced to molybdenum blue, which was measured spectrophotometrically at 750 nm using a Shimadzu Spectrophotometer. Lipid peroxidation levels in stomach homogenates were assayed by the method of Ledwozyw et al. (Ledwozyw et al. 1986). In brief, the adducts formed following boiled tissue homogenate with thiobarbutiric acid was extracted with n-butanol. The difference in optical density at 532 nm was measured in terms of the liver malondialdehyde (MDA) content, also of thiobarbituric acid reactant substances (TBARS), which was undertaken as an index of lipid peroxidation. The results were expressed as nmol MDA/mg protein. Stomach glutathione (GSH) levels were measured according to Beutler’s method using Ellman’s reagent (Beutler 1975). The procedure was based on the reduction of Ellman’s reagent by SH groups to form 5,5°-dithiobis(2-nitrobenzoic acid) with an intense yellow color, measured spectrophotometrically at 412 nm using a Shimadzu Spectrophotometer. The results were expressed as nmol GSH/mg protein. The protein content in the supernatants was estimated by the method of Lowry using Bovine serum albumin as standart (Lowry et al. 1951).

Statistical Analyses

The results were evaluated using an unpaired t test ANOVA variance analysis using the NCSS statistical computer package.

RESULTS

The fundus tissues from rats administered only the antioxidants had the same microscopic appearance as the intact control group. The mucosal surface was covered with the mucus dyed with PAS in all groups. In the group treated with cadmium only, the discontinuity of mucus in surface were observed as compared to the other group. Also, the discontinuity and degeneration in the surface epithelium dyed with Masson’s trichrome were observed in rats exposed to cadmium, as compared to control groups. In the group treated with cadmium only, distensions in the lumen of some fundus glands were greater than those of the other groups. The degenerative changes were reduced in the group administered vitamin E + vitamin C + selenium + Cd. Immunoreactive ghrelin cells were observed scattered all through the mucosa, prominently at the base of the fundus glands. Occasionally, ghrelin-immunoreactive cells were found among surface epithelial cells. Metallothionein-immunoreactive cells were mainly localized all through the mucosa but were rarely found in the middle region of the fundus glands. There was no significant change in ghrelin and metallothionein-immunoreactive cells in fundus mucosa (Figure 1).

The mean serum phospholipid and total lipid levels are given in Table 1. There were notable differences in the serum phospholipid and total lipid levels in all four groups (p _ANOVA= 0.0001). Serum phospholipid and total lipid levels in cadmium group were significantly increased when compared to the control group (p _{t test} = 0.0001). Vitamin C, vitamin E, and selenium administration caused remarkable decreases in serum phospholipid and total lipid levels (p _{t test} = 0.001; p _{t test} = 0.0001) in cadmium group. The mean stomach GSH and LPO levels of the four groups are given in Table 2. As seen in Table 2, a meaningful difference in the stomach GSH and LPO levels of the four groups was observed (p _ANOVA = 0.0001). Stomach LPO levels in cadmium group were significantly increased when compared to the control group (p _{t test} = 0.0001). The administration of vitamin C, vitamin E, and selenium caused a significant increase in LPO levels in the cadmium group (p _{t test} = 0.0001). In the cadmium group, stomach GSH levels were lower than those of the other groups (p _ANOVA = 0.0001). The administration of vitamin C, vitamin E, and selenium to rats seemed to have caused an increase in the stomach GSH levels in cadmium groups (p _{t test} = 0.003).

DISCUSSION

In the cadmium group, the discontinuity of mucus and degeneration in the surface epithelium were observed as compared to the other group. A breakdown in the structure of mucosa reflects cadmium toxicity. Metallothionein can be used as a biomarker of metal exposure. Both GSH and metallothionein are known to act as scavengers of ROS. The presence of cellular metallothionein reduces the adverse effects of cadmium (Klaassen, Liu, and Choudhuri 1999). Shimizu and Morita (1990) reported that GSH is utilized for metallothionein synthesis in response Cd. The synthesis of metallothionein in the liver increased after the administration of vitamin C (Onosaka et al. 1987). The hepatoprotective effect of metallothionein was observed in hepatocyte cultures from metallothionein transgenic mice. The increased cytosolic Cd was bound primarily to metallothionein, and metallothionein shows a protective effect against Cd hepatotoxicity (Liu et al. 1995). Metallothionein-null and wild-type mice showed similar cadmium absorption and tissue distribution following oral cadmium administration (Liu, Liu, and Klaassen 2001). On the other hand, Singh and Rana (2002) reported that antioxidants and metallothionein employ different mechanisms of protection against Cd toxicity, in accordance with our findings. Ghrelin exerts a potent protective action on the stomach of rats exposed to ethanol or restraint stress (Brzozowski et al. 2005). Endogenous antioxidants, such as ghrelin, attenuate the oxidative-stress response (Dong and Kaunitz 2006). In this study, a difference was not found in the ghrelin cells of fundus mucosa against cadmium toxicity. It is suggested that ghrelin cells may not play a role in the toxicity of cadmium.

The harmful effects of ROS are balanced with the protective effects of antioxidants. Although vitamin C is well known as an antioxidant in animal tissues, some literatures indicates that an increased intake of vitamin C is associated with a reduced risk of toxicity (Grosicki 2004; Shiraishi, Uno, and Waalkes 1993). It is reported that vitamin C in plasma increases resistance to lipid peroxidation according to dosage (Suh, Zhu, and Frei 2003). Vitamin C is a powerful antioxidant along with vitamin E. Pretreatment with vitamin E exhibited a protective role against the toxic effects of cadmium on enzymatic and nonenzymatic components of antioxidant defense system (Ognjanović et al. 2003). Stimulation of lipid peroxidation and decrease in GSH level after Cd were observed in rat fundus. Many studies have shown that cadmium induces lipid peroxidation (Jurczuk et al. 2004; Manca et al. 1991; Sarkar et al. 1995). Markedly increased levels of lipid peroxidation markers and a significant decrease in vitamin C and vitamin E were observed in cadmium intoxicated rats (Pari et al. 2007). It is reported that enhanced production of ROS results in increased lipid peroxidation (Stohs, Bagchi, and Hassoun 2001). Cadmium induces oxidative stress in tissues by increasing lipid peroxidation (El-Demerdash et al. 2004; El-Sharaky et al. 2007). However, the prooxidant state induced by cadmium in the liver does not appear to be imputable to lipid peroxidation (Casalino, Sblano, and Landriscina 1997). The present study shows a linear relationship between antioxidants and lipid peroxidation. It was reported that selenium provided protection against Cd-induced lipid peroxidation (Yiin et al. 1999). Ognjanović et al. (2007) reported that selenium may ameliorate Cd-induced oxidative stress by decreasing lipid peroxidation and altering antioxidant defence system in the livers and kidneys of rats. In addition, the antioxidative protection does not seem to be associated with ghrelin and metallothionein-immunoreactive cells in fundus mucosa. The administration of antioxidants had a protective effect to some extent on stomach of the group given vitamin E + vitamin C + selenium + Cd, because antioxidants do not have a protective effect against lipid peroxidation.

In the cadmium group, stomach GSH levels were lower than those of the other groups. The observed decrease in GSH might be a result of Cd administration in the scavenging of free radicals. Stohs, Bagchi, and Hassoun (2001) reported that cadmium depletes glutathione and protein-bound sulfhydryl groups. El-Maraghy et al. (2001) reported that after repeated administration of Cd, tissue Cd accumulation was accompanied by increased hepatic and renal GSH levels. Intracellular glutathione levels are related to vitamin E protection against oxidation-induced cell damage (Rana and Verma 1996). They demonstrated that selenium protection is affected by changes in Cd- and glutathione-dependent enzymes. It is reported that selenium exerted a protective effect against the toxicity of cadmium (Lindh, Danersund, and Lindvall, 1996). Selenium could partially alleviate the oxidative stress induced by cadmium in rat kidney (Xiao et al. 2002). Exogenous selenium ions are readily incorporated into cell organelles and cytosol where they inhibit lipid peroxidation in the nuclei and microsomes (Guseinov, Nasibov, and Dzhafarov 1990). The total lipid levels change after cadmium treatment in kidney tissues of rats, whereas pretreatment of rats with selenium protects against these changes resulting from cadmium administration (El-Sharaky et al. 2007). The present study shows that our combined antioxidant treatment protects the stomach tissues against the toxicity of cadmium. In conclusion, the administration of cadmium in combination with vitamin E + vitamin C + selenium partially prevents cadmium-induced oxidative damage in the stomach.