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Article Excerpt
SEVERAL XENOBIOTICS, SUCH AS ALCOHOLS, for example, are increasingly recognized as substances that disturb cellular metabolism, including redox balance. In most cases, it is connected with metabolism of these xenobiotics. Ethanol is metabolized into acetaldehyde and then into acetate mainly in the liver, and this process is accompanied by formation of free radicals. It has been shown that chronic ethanol intoxication enhances the generation of mainly superoxide radical and hydrogen peroxide (Kukielka and Cederbaum, 1994). Enhanced generation of superoxide radical is caused by an increase in the level of NADH (reduced form of the nicotinamide adenine dinucleotide [NADI) produced during oxidation of ethanol as well as its metabolite (i.e., acetaldehyde) (Sochman, 1994). A decrease in the NAD/NADH ratio causes conversion of xanthine dehydrogenase into xanthine oxidase, an enzyme responsible for generation of superoxide radical (Kato et al., 1990). This transformation also occurs during acetaldehyde oxidation into acetate (Puntarulo and Cederbaum, 1989). The increase in NADH concentration is also responsible for enhanced release of iron (II) ions from ferritin (Shaw et al., 1988). An increase in free iron (II) ions, which catalyze free radical reactions, leads to an increase in the level of reactive oxygen species (ROS) observed in ethanol intoxication (Shaw et al., 1988). Chronic alcohol intoxication is also accompanied by a decrease in the activities of ethanol-metabolizing enzymes (i.e., alcohol and aldehyde dehydrogenases), and consequently, by an increase in acetaldehyde accumulation. In this situation, xanthine oxidase may also catalyze superoxide radical generation using acetaldehyde as a substrate (Kellogg and Fridovich, 1975). Moreover, increased generation of oxygen and ethanol-derived free radicals has been observed at the microsomal level, especially through the application of the ethanol-inducible cytochrome P450 isoform (Krikun et al., 1984). A decrease in antioxidant status during ethanol intoxication caused the generation of oxidative stress (Kurose, 1996). This oxidative stress results in enhanced lipid peroxidation and changes in the structure and function of other important cellular components such as protein and DNA (Rouach et al., 1997; Wang et al., 1990).
Therefore, scientists have searched for potent antioxidants, especially among natural products. One such potentially health-promoting beverage is tea. It was generally believed that only green tea prepared by dehydration of Camellia sinensis leaves, which contain monomeric polyphenols, possesses antioxidant properties (Graham, 1992). Recent investigations, however, indicate that black tea obtained by fermentation of tea leaves and containing only a small amount of monomeric polyphenols (catechins) and multimeric polyphenols, (theaflavins [TFs], and thearubigins)--whose biological activities are less documented but still extensively examined--also reveals antioxidant abilities (Frei and Higdon, 2003; Graham, 1992). As a result, black tea has been proved to protect against cancer progression and heart diseases (Dufresne and Farnworth, 2001 ; Yang et al., 2003; Rietveld and Wiseman, 2003). However, the biological activity of black tea as a source of antioxidants requires further investigation.
The purpose of this study was to investigate potential protective effects of black tea on the consequences of ethanol-induced oxidative stress in the liver manifested by lipid and protein modifications.
Method
Black tea
Black tea--Camellia sinensis (Linnaeus) O. Kuntze (standard research blends--lyophilized extract)--was provided by TJ Lipton (Englewood Cliffs, NJ) and was dissolved in drinking water at concentration of 3 g/1. Tea was prepared three times per week and stored at 4[degrees] C until use. The content of drinking vessels was renewed every day. Black-tea extract contained catechins (epigallocatechin gallate [EGCG]: 14.53 mg/1; epigallocatechin [EGC]: 2.21 mg/1; and epicatechin [EC]: 2.83 mg/1) and YFs (theaflavin [[TF.sub.1]]; theaflavin 3-gallate [[TF.sub.2]A]; theaflavin 3'-gallate [[TF.sub.2]B]; and theaflavin 3, 3'-digallate [[TF.sub.3]] in the amount of 156.16 mg/g dried extract for all four TFs). The levels of catechins and TFs were determined by modified high-performance liquid chromatography (HPLC) methods of Mattila et al. (2000) and Lee et al. (2000).
Animals
Two-month-old male Wistar rats were used for the experiment. They were housed in groups with free access to a granular standard diet and water and were maintained under a normal light-dark cycle. All experiments were approved by the Local Ethics Committee in Biatystok, Poland, referring to the Polish Act Protecting Animals of 1997.
The animals were divided into the following groups:
* The control group was treated intragastrically with 1.8 ml of physiological saline every day for 4 weeks (n = 6).
* The black-tea group was given black-tea solution ad libitum instead of water for 1 week. Then, the group was treated intragastrically with 1.8 ml of physiological saline and received black-tea solution ad libitum instead of water every day for 4 weeks (n = 6).
* The ethanol group was treated intragastrically with 1.8 ml of ethanol in doses increased by ethanol concentration from 2.0 to 6.0 g/kg body weight every day for 4 weeks. The dose of ethanol was gradually increased by 0.5 g/kg body weight every 3 days (n = 6).
* The ethanol-and-black-tea group was given black-tea solution ad libitum instead of water for 1 week. Next, it was treated intragastrically with 1.8 ml of ethanol in doses increased by ethanol concentration from 2.0 to 6.0 g/kg body weight and received black-tea solution ad libitum instead of water every day for 4 weeks.
The animals were deprived of food 12 hours before being intubated and then received saline or ethanol solution intragastrically by gavage.
The amount of black tea given to rats was an amount equivalent to 40-50 cups of tea daily (approximately 100 mg/kg body weight). However, this amount was necessary to achieve the therapeutic effect.
Preparation of tissue
After the above procedure, the rats were sacrificed under ether anesthesia (six animals in each group). The livers were removed quickly and placed in iced 0.15 M NaCl solution, perfused with the same solution to remove blood cells, blotted on filter paper, weighed and homogenized in 9 ml ice-cold 0.25 M sucrose and 0.15 M NaC1 with the addition of 6 [micro]l 250 mM butylated hydroxytoluene (BHT) in...
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