Abstract: The aim of this research was to examine the effects of (+)-catechin on the levels of Malondialdehyde (MDA), homocysteine, vitamin C, cholesterol, GSH, GSSG and lipophilic vitamins in serum and erythrocytes of old and ovariectomized rats after exposure to the carcinogen potassium bromate. Female Wistar rats (n = 30) were randomly divided into 3 groups. The 1st group was used as a control, the 2nd group was treated with KBrO₃ and 3rd group received C + KBrO₃. Rats in the KBrO₃ and C + KBrO₃ groups were injected intraperitoneally with a single dose of KBrO₃ (80 mg kg^-1) in physiological saline. After 2 days, those in the C + KBrO₃ group were intraperitoneally injected with catechin (30 mg kg^-1) 4 times week^-1. Physiological saline was injected into the control group rats. The results indicate that the serum cholesterol level in the KBrO₃ group was higher than in the other 2 groups (p<0.01). MDA level was lower in the C + KBrO₃ group than the others (p<0.001). The amount of α-tocopherol increased in the KBrO₃ treated groups (p<0.05, p<0.01). Vitamins C and D₂ levels were higher in the KBrO₃ and C + KBrO₃ groups than the control group (p<0.001). In erythrocytes, the cholesterol level decreased in the KBrO₃ group and the vitamin D₃ level decreased in the C + KBrO₃ group (p<0.005). MDA level in the KBrO₃ group was higher than the control and C + KBrO₃ group (p<0.05). GSH, GSSG and α-tocopherol levels were lower in erythrocytes from the KBrO₃ and C + KBrO₃ groups than the control group (p<0.001). The GSH/GSSG ratio was high in the KBrO₃ group; there was no difference between the control and C + KBrO₃ groups. In conclusion, it can be said that administration of catechin decreased MDA in the serum and erythrocytes of old and ovariectomized Wistar rats and prevented the decrease of erythrocyte cholesterol.

INTRODUCTION

Flavonoids are polyphenolic antioxidants naturally present in vegetables, fruits and beverages such as tea and wine. They have been shown to have potential for protecting against chronic diseases and atherosclerosis. They can act as free radical scavengers and transient metal ion chelators. Catechin has pharmacological effects such as anticarcinogenic, antioxidant, dental and antimicrobial activities and is active in preventing cardiovascular disease (Kang et al., 1999; Demeule et al., 2000). The pure catechins to be more powerful antioxidants than vitamins C, E and β-carotene in an in vitro lipoprotein oxidation model (Joe et al., 1998).

Catechins have also been shown to protect neurons, enhance resistance of red blood cells to oxidative stress, inhibit ultra-violet radiation-induced oxidative stress in skin and reduce the blood cholesterol level by protecting low density lipoproteins from oxidation (Chu et al., 2004). However, catechins have been reported to enhance colon carcinogenesis in rats, suggesting that they may have carcinogenic as well as anticarcinogenic effects (Furukawa et al., 2003). These compounds show antithrombic activity, which might be due to an antiplatelet effect rather than anticoagulation (Son et al., 2004). Catechins, have been considered potential chemotherapeutic agents since they inhibit carcinogenesis in several animal models (Hirose et al., 2001). The protective activity of catechins is generally attributed to the free radical-scavenging (Kang et al., 1998).

Potassium Bromate (KBrO₃) is an oxidizing agent that has been reported to be carcinogenic in rats, causing renal tumours (Chipman et al., 1998; Cadenas and Barja, 1999).

It increases the level of 8-oxodeoxyguanosine (8-OH-deoxyguanosine, 8-oxodG) in the kidney DNA of treated rats. In female rats, ovariectomy artificially induces a marked reduction in endogenous estrogen concentrations subsequently causing a negative bone remodeling balance that augments bone loss and increases the incidence of osteopenia (Wronski et al., 1988). Estrogens influence the growth and functioning of female and male reproductive tissues, maintain the skeletal and central nervous system, provide cardioprotective effects in the cardiovascular system and protect against colon cancer and aging skin (Ososki and Kennelly, 2003). Plant-derived phytoestrogens are compounds structurally or functionally mimic mammalian estrogens and therefore, are considered to play an important role in the prevention of cancers, heart disease, menopausal symptoms and osteoporosis (Setchell et al., 1998; Adlercreutz, 2002; Kronenberg and Berman, 2002). The aim of this study was to examine the effects of (+)-catechin on the levels of MDA, homocysteine, vitamin C, cholesterol, GSH, GSSG and lipophilic vitamins in serum and erythrocytes from old and ovariectomized female Wistar rats exposed to potassium bromate (KBrO₃).

MATERIALS AND METHODS

Animals: A total 32 old ovarectomized female Wistar rats were used in this study. The animals were housed in cages where they had ad libitum rat chow and water in an air-conditioned room with a 12 h light/dark cycle and were randomly divided into 3 groups. The 1st group was used as a control, the 2nd group received potassium bromate (KbrO₃) and 3rd group received C + KBrO₃. Rats in the KBrO₃ and C + KBrO₃ groups were injected intraperitoneally with a single dose of potassium bromate (80 mg kg^-1) in physiological saline. Two days later, the rats in C + KBrO₃ group were intraperitoneally injected with (+)-catechin (30 mg kg^-1) 4 times week^-1. Physiological saline was injected into the control group rats. These treatments were continued for 5 weeks, then each rat was decapitated and blood samples were collected and stored in -85°C prior to biochemical analysis.

Sample preparation for biochemical analyses: Blood samples were collected into tubes. Blood was centrifuged at 2, 500xg at 4°C for 10 min and serum was separated. The erythrocyte pellet was washed 3 times with 0.9% NaCl and centrifuged for 5 min at 2.500xg after each wash. The pellet and serum samples were prepared freshly for each biochemical analysis.

Determination of erythrocyte glutathione: GSH and GSSG were separated and analyzed by fully automatic High Performance Liquid Chromatography (HPLC). The HPLC equipment consisted of a pump (LC-10 ADVP), a UV-visible detector (SPD-10AVP), a column oven (CTO-10ASVP), an autosampler (SIL-10ADVP), a degasser unit (DGU-14A) and a computer system with class VP software (Shimadzu, Kyoto, Japan). A discovery RP-Amide C16 column (150x4.6 mm, 5 μm; Sigma, USA) was used and the mobile phase was 50 mM NaClO₄ + 0.1% H₃PO₄ (Serada et al., 1997) at a flow rate of 1 mL min^-1. Samples were detected at 215 nm with the column oven set at 40°C. Briefly, a 300 mg erythrocyte pellet was homogenized in 2 mL, 5 mM EDTA, 50 mM NaClO₄, 0.1% H₃PO₄ buffer. Proteins were precipitated by adding 500 μL of 10% metaphosphoric acid (Yoshida, 1996). The samples were vortexed for 20 sec and centrifuged at 8000xg for 10 min at 4°C. The supernatants were transferred to autosampler vials for HPLC. Quantification was carried out by external standardization using Class VP software. The results were expressed as μg g^-1 wet cell pellet.

Determination of lipid-soluble vitamins in serum and erythrocytes: Serum samples (200 μL) or 300 mg erythrocyte pellets were homogenized in 3 mL acetonitrile/ methanol (3.1, v v^-1) and the samples were vortexed for 30 sec and centrifuged at 6000xg for 10 min at 4°C. Supernatants were transferred to autosampler vials for HPLC. Acetonitrile/methanol (75.25, v v^-1) was used as the mobile phase at a flow-rate of 1 mL min^-1. The temperature of the analytical column was kept at 40°C. A Supelcosil™ LC 18 DB column (250x4.6 mm, 5 μm; Sigma, USA) was used. Retinol (vitamin A) and retinol acetate were detected at 320 nm and vitamins D₂, D₃ and K₁, α- tocopherol, α-tocopherol acetate and α-tocopherol at 215 nm. Individual vitamins were identified by frequent comparison with authentic external standard mixtures analyzed under the same conditions. Quantification was carried out by external standardization using Class VP software. The results were expressed as μg g^-1 wet cell pellet for erythrocytes and μg dL^-1 for serum.

Cholesterol in serum and erythrocytes: Serum samples (200 μL) or 300 mg erythrocyte pellets were extracted in 3 mL acetonitrile/isopropyl alcohol (70:30, v v^-1) and the mixtures were vortexed for 30 sec and centrifuged at 6000xg for 10 min at 4°C. Supernatants were transferred to autosampler vials for HPLC. Acetonitrile-isopropanol (70:30 v v^-1) was used as mobile phase at a flow rate of 1 mL min^-1 (Bragagnolo and Amaya, 2003). A Supelcosil LC 18™ DB column (250x4.6 mm, 5 μm) was used. Samples were detected at 202 nm and the column oven was set at 40°C (Katsanidis and Addis, 1999). Quantification was carried out by external standardization using class VP software. The results were expressed as μmol g^-1 wet cell pellet for erythrocytes and μmol dL^-1 for serum samples.

Vitamin C and MDA: Serum samples (200 μL) or 300 mg erythrocyte pellets were homogenized in 2.5 mL of 5 mM 1-hexane sulphonic acid sodium salt + 0.1 H₃PO₄ buffer. Proteins were precipitated by adding 0.5 mL of metaphosphoric acid (5% w v^-1) and the samples were centrifuged at 10,000 g for 5 min. The supernatants were transferred to autosampler vials for HPLC. A Discovery RPAmide16 (150x4.6 mm, 5 μm, Sigma, USA) column was used. The mobile phase was 5 mM 1-hexane sulphonic acid sodium salt + 0.1 H₃PO₄ buffer with acetonitrile (90 + 10%) at a flow rate of 1 mL min^-1. Samples were detected at 244 nm and the column was maintained at 37°C. Quantification was carried out by external standardization using class VP software. The results were expressed as nmol for MDA and μg for VC.

RESULTS

The serum cholesterol level was increased in the KBrO₃ group (p<0.01) but there was no difference between the control and C + KBrO₃ groups (p>0.05). MDA was decreased in the C + KBrO₃ group but there was no difference between the control and KBrO₃ groups (p>0.05). The α-tocopherol levels in the KBrO₃ and C + KBrO₃ groups were higher than the control (p<0.05, p<0.01, respectively). The α-tocopherol acetate, δ-tocopherol, retinol and vitamin D₃ levels did not differ among the groups (p>0.05).

Table 1:	Biochemical parameters in the serum samples

Table 2:	Biochemical parameters in the erythrocytes
Values are the mean±SEM; ANOVA: one-way ANOVA p-value; a: p>0.05, b: p<0.05, c: p<0.01, d: p<0.001 using the post hoc Fischer’s LSD test

However, the vitamin C and D₂ levels in the KBrO₃ and C + KBrO₃ groups were higher than in the control group (p<0.001) (Table 1).

In the erythrocytes; GSH and GSSG were lower in the KBrO₃ and C + KBrO₃ groups than the control group (p<0.05). However, the GSH/GSSG ratio was high in the KBrO₃ group; there were no differences between the control and C + KBrO₃ groups Cholesterol did not differ between the control and C + KBrO₃ groups but was lower in the KBrO₃ group (p<0.05). MDA were increased in the KBrO₃ group (p<0.05), but there was no difference between the control and C + KBrO₃ groups (p>0.05) The vitamin D₃ level was lower in the C + KBrO₃ group than the KBrO₃ and control groups (p<0.05). α-tocopherol acetate and α-tocopherol were lower than control levels in the KbrO₃ and C + KBrO₃ groups (p<0.001). Vitamin K₁ was the same in the control and C + KBrO₃ groups and decreased in the KBrO₃ group (p<0.01) (Table 2).

DISCUSSION

Lipid peroxidation products are involved in some of the pathophysiological effects associated with oxidative stress in cells and tissues (Ostrowska et al., 2004; Komatsu and Hiramatsu, 2000). Aldehydes produced by lipid peroxidation are rather long-lived and can therefore, diffuse from the site of their origin to attack intracellular and extracellular targets (Esterbauer et al., 1991). They disrupt important structural and protective functions associated with biomembranes in various pathological events in vivo (Barclay, 1993).

We found that the MDA level in the serum and erythrocytes of KBrO₃ group were than the control and C + KBrO₃ groups. It has been reported that catechins suppress the formation of lipid peroxidation products in tissues and subcellular fractions (Ostrowska et al., 2004). Glutathione and MDA levels have been commonly used to assess antioxidant defenses and lipid peroxidation. MDA has also been extensively used to evaluate the extent of tissue and cell damage through lipid peroxidation (Mejia and Mares, 2002). Many transition metalions are also able to break down lipid hydroperoxides to trigger free radical-mediated lipid peroxidation (Aust et al., 1985). Therefore, the oxidative breakdown of the membrane polyunsaturated fatty acids is accompanied by the formation of secondary products. These compounds are highly reactive and may act as secondary toxic messengers of the primary free radical event (Mitchell and Petersen, 1997). In consequence, the probability of cell injury may increase (Tuma et al., 1987). Aldehydes generated during lipid peroxidation also form glutathione conjugates, leading to a significant decrease in cellular glutathione concentration, which may partly account for the cytotoxicity of oxidative stress (Kinter and Roberts, 1996).

Lipid peroxidation is a degradative process causing functional abnormalities and pathological changes that lead to and exacerbate myriad chronic diseases (Willcox et al., 2003). Catechins may decrease the concentration of lipid free radicals and terminate the initiation and propagation of lipid peroxidation (Ostrowska et al., 2004). Because catechin derivatives are water-soluble antioxidants, they could reduce the mobility of the free radicals into the lipid bilayer as well.

In our results, the α-tocopherol level was higher in the C + KBrO₃ group than the control group. Catechins prevent the loss of the lipophilic antioxidant α-tocopherol by repairing tocopheryl radicals and by protecting the hydrophilic antioxidant ascorbate, which also repairs this radical (Ostrowska et al., 2004). Α-tocopherol is a major antioxidant in the plasma or serum for protection of low-density lipoproteins (Zhu et al., 1999). α-tocopherol scavenges active oxygen radicals in biological systems and protects unsaturated lipid from peroxidation, producing the α-tocopheroxyl radical (Mukai et al., 2005). Catechins may counteract a decrease in α-tocopherol by acting as antioxidants in vivo (Nanjo et al., 1993).

The present study has shown that the serum cholesterol level in the KBrO₃ group was higher than in the control and C + KBrO₃ groups. Oxidized cholesterol has various deleterious actions such as cytotoxicity, carcinogenicity, atherogenicity, inhibition of DNA synthesis, suppression of immune function and modulation of lipid metabolism both in vitro and in vivo. It has been reported that catechin inhibits cholesterol oxidation in lipoproteins through its radical scavenging action (Loest et al., 2001).

A correlation has been reported between the antiproliferative potency of flavonoids and their ability to inhibit the cellular accumulation of vitamin C: thus, quercetin and genistein are strongly antiproliferative and affect ascorbic acid accumulation. The inhibition is dose-dependent and can be observed after as little as 10 min incubation. Flavonoids with little antiprolifeative effect, naringenin and catechin, have little effect on ascorbic acid accumulation. Therefore, the antiproliferative properties of these molecules could be linked to their capacity to affect ascorbate accumulation (Osada et al., 2001). In our study, catechin partly inhibited the accumulation of vitamin C in serum.

Glutathione (GSH) level was low in both the KBrO₃ and C + KBrO₃ groups. GSH plays a key role in protecting cells against electrophiles and free radicals and it is a substrate or a cofactor for a number of protective enzymes such as GSHpx, the GSH S-transferases and glyoxalase (Kuo et al., 1997). In addition, GSH has several vital roles including antioxidation, maintenance of the intracellular redox state and modulation of the immune response and detoxification of xenobiotics (Navarro et al., 1997). With respect to cancer, glutathione metabolism can play both protective and pathogenic roles. It is crucial in the removal and detoxification of carcinogens and alterations in this pathway can have a profound effect on cell survival. However, by conferring resistance to a number of chemotherapeutic drugs, elevated levels of glutathione protect tumor cells in bone marrow, breast, colon, larynx and lung (Kuo et al., 1997). Reduced GSH can directly scavenge radicals and peroxides via mixed disulfide formation, or upon oxidization to GSSG. Various oxygen radical stresses deplete GSH and form GSSG in the short term (Kuo et al., 1997). In particular, a decreased mitochondrial GSH pool has been reported in rats and this may play an important role in pathogenesis (Balendiran et al., 2004). Catechins and gallic acid are poor inhibitors of GSH reductase, possibly owing to the absence of key structural features from the molecules. Inhibition of GSH-reductase by plant polyphenols and the consequent low level of reduced GSH may impair the detoxification of anticancer drugs by GSH conjugation and sensitize drug-resistant cells to chemotherapeutic agents (Lu, 1999). GSH is depleted by KBrO₃, suggesting an interaction (Chipman et al., 1998). Our results show that the decrease in erythrocyte GSH in the KBrO₃ treated groups was not prevented by catechin.

CONCLUSION

In conclusion, MDA levels in the sera and erythrocytes of ovariectomized Wistar rats were decreased by catechin administration. In addition, decrease of erythrocyte cholesterol was prevented by catechin treatment. However, the erythrocyte GSH levels were low in both the catechin and KBrO₃ treated groups.

ACKNOWLEDGEMENT

This research was supported by the State Planning Organization of the Turkish Republic, under grant numbers DPT-2002K120240 and 2003K120440.