* Department of Tea Science, Zhejiang University, Hangzhou, China 310029; and
Laboratory of Visual Information Processing, Research Centers of Brain and Cognitive Science, Institute of Biophysics, Academia Sinica, 15 Datun Road, Chaoyang District, Beijing, China 100101
Received April 22, 2002; accepted June 16, 2002
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ABSTRACT |
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Key Words: tea; catechins; lead toxicity; lipid peroxidation; antioxidants; oxidative stress; ESR spin labeling; membrane fluidity.
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INTRODUCTION |
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A growing amount of evidence indicates that cellular damage mediated by reactive oxygen species (ROS) may be involved in the pathology associated with lead intoxication (Bechara et al., 1993; Hermes-Lima et al., 1991
). The malondialdehyde levels in blood were strongly correlated with lead concentration in the blood of exposed workers (Jiun and Hsien, 1994
). In erythrocytes from the workers exposed occupationally to lead, the activities of the antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase, were remarkably higher than that in non-exposed workers (Monteiro et al., 1985
). Gurer et al. demonstrated that lead increased the prooxidant/antioxidant ratio in a concentration-dependent manner in lead-treated CHO cells and in rats (Gurer et al., 1999
). The results suggest that antioxidants might play an important role in the treatment of lead poisoning.
Tea, including black, green, and oolong tea, is one of the most widely consumed beverages in the world. During the last decade, numerous in vitro and in vivo studies had suggested that tea and tea polyphenols had strong antioxidant activity (Guo et al., 1996, 1999
; Shen et al., 1993
), and had numerous potentially beneficial medicinal properties including inhibition of carcinogenesis, tumorigenesis, and mutagenesis, as well as the inhibition of tumor growth and metastasis (Yang et al., 1993). The major polyphenolic compounds in tea are catechins. The four most abundant naturally occurring tea catechins, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) are shown in Figure 1
.
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Tea is a kind of excellent scavenger of free radicals and chelator of heavy metal (Guo et al., 1991; Kumamoto et al., 2001), but whether tea catechins have protective effects on oxidative stress after lead treatment remains unclear. The present study showed tea catechins could reduce the toxicity of lead in HepG2 cells by examination of the effect of lead on cell viability, malondialdehyde (MDA) levels, and cell-membrane fluidity in the presence or absence of different kinds of tea catechins.
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MATERIALS AND METHODS |
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Cell culture.
The human hepatocellular carcinoma cell line HepG2 retains many parenchymal cell functions. It has been shown that it is useful for evaluations of the mechanism of toxicity (Borenfreund et al., 1990; Marinovitch et al., 1988
). HepG2 cells were grown as monolayer cultures in DMEM, supplemented with 10% heat-inactivated newborn calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 1% non-essential amino acid, and 2 mM glutamine. Only cells in exponential growth were used for the experiments.
Cells were grown at 37°C in disposable plastic bottles (Nunc, USA) in a humidified atmosphere of 5% CO2, 95% air. The medium was replaced twice a week, and cells were trypsinized and diluted every 7 days at a ratio of 1:3.
Cell viability assay.
In the experiments, 1 x 104 cells were plated in each well of 96-well plates, and were allowed to attach to the substrate for a 24-h period. Cells were exposed to lead for an additional 24 h in the absence or presence of different concentrations of tea catechins. Cell viability was determined using the MTT assay (Mosmann et al., 1983). In brief, 20 µl of 5 mg/ml MTT in PBS was added to each well and the plates incubated at 37° for a further 4 h. The media were then removed and the purple formazan crystals dissolved in 150 µl DMSO. The absorbency of each well was then measured at 570 nm with a Bio-RAD 3350 microplate reader, and the percentage viability was calculated.
Measurement of TBARS.
Lipid peroxidation was assayed by determining the production rate of thiobarbituric acid reactive substances (TBARS) and was expressed as malondialdehyde (MDA) equivalents. In brief, cells grown on 6-well plates were washed with 0.01 M PBS, scraped, and resuspended in 1 ml PBS. An aliquot was taken out for a protein assay, and 0.5 ml TBA reagent (100 mg trichloroacetic acid, 3.35 mg thiobarbituric acid) was added to each tube and vortexed. The reaction mixture was incubated at 90°C for 20 min and stopped on ice. After cooling to room temperature, TBARS were extracted with 1.0 ml n-butanol and separated at 3000 x g centrifugation for 5 min. The absorbency of the total TBARS was measured at 532 nm. Tetraethoxypropane in absolute ethanol was used to prepare MDA standards. The measurements were performed in triplicate and the results were expressed as nmol equivalent of MDA/mg protein.
Spin labeling the cells with 5-doxyl or 16-doxyl.
Fatty acid spin-labels of 5-doxyl and 16-doxyl, which have a stable nitroxide radical ring at the C-5 and C-16 positions, respectively, were used as a lipid probe in the cell membrane. They are well dissolved in lipids and their ordering and dynamics reflect the motion of the surrounding phospholipid hydrocarbon chains. In brief, 100 µl HepG2 cell suspension (107 cells/ml) was mixed with 5 µl of 5-doxyl or 16-doxyl (1.0 mM) spin label, incubated at 37° for 60 min, then the free labels washed out by 0.01 M PBS from the cell system until there were no ESR signals in the supernatant. ESR measurement condition: microwave power 20 mW, modulation amplitude 0.2 mT, X-band, modulation frequency 100 KHz, sweep width 10 mT, and temperature 298K.
Membrane fluidity calculation.
The membrane fluidity characteristics were estimated from the line width and shape of the ESR spectra. Lower order and faster motion means higher membrane fluidity. The order of membrane hydrocarbon chains is described by the order parameter (S) and their motion is described by the rotational correlation time (c). They are defined as follows (Juntao et al., 2001
):
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where h(0), h(1), and h(-1) are the peak height of the center, low, and high field lines, respectively; H(0) is the width of the central line; and A|| and A
are parallel and perpendicular hyperfine splitting parameters of the spectrum, respectively, as shown in Figure 2
.
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RESULTS |
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DISCUSSION |
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Tea catechins are strong scavengers against superoxide, hydrogen peroxide, hydroxy radicals, and nitric oxide produced by various chemicals. They also could chelate with metals because of the catechol structure (Rice-Evans et al., 1997). These characteristics make tea catechins ideal candidates for treatment of lead toxicity. The data from our studies of HepG2 cells indicated that the higher concentration of lead treatment decreased cell viabilities and increased lipid peroxidation levels. Treatment by tea catechins increased cell viability and reversed the effects of lead on oxidative stress parameters in a concentration-dependent manner. The galloylated catechins showed stronger protective effect against oxidative damage than that of nongalloylated catechins, which is similar to the result of scavenging ability on free radicals (Guo et al., 1996
, 1999
). Galloylated catechins containing more phenolic hydroxyl groups had stronger chelating ability with metal ions than nongalloylated catechins (Guo et al., 1991). Therefore, the protective effect of tea catechins on oxidative damage in HepG2 cells exposed to lead might be related to both their ability to scavenge free radicals and to chelate metal ions.
ESR spin labeling technique is a sensitive and reliable method to study the physical state of cell membranes. Order parameter (S) and rotational correlation time (c) represent the degree of hydrocarbon chains long-range alignments along the membrane and the motion state of these chains. As shown in Figure 7
, the increase of lipid peroxidation levels indicated that lead caused oxidative damage to hepatic cell membranes. The peroxidation of hepatic cell membrane phospholipids and accumulation of lipid peroxides are expected to modulate the membrane fluidity and consequently the membrane function. The observed changes in the rotational correlation time (
c) and order parameter (S) (Fig. 10
) indicated that the fluidity near the surface of the membrane was decreased after 100 µM-lead treatment, but the fluidity in the hydrophobic core of the membrane was not affected after the treatment. Lead induced arachidonic acid augmentation (Lawton et al., 1991) and bound strongly to phosphatidylcholine membranes in vitro (Shafiqur-Rehman et al., 1993), which could result in altered membrane integrity, permeability, and fluidity. These might be connected with the enhanced lipid peroxidation in HepG2 cells.
Tea catechins are mainly composed of 5060% EGCG, 812% EGC, 1520% ECG, and 47% EC. As reported previously, tea catechins scavenged free radicals in the order: EGCG ECG > EGC > EC (Guo et al., 1996
, 1999
). Okabe also reported the similar order in inhibiting growth of human lung cancer cell line PC-9 (Okabe et al., 1997
). Because of its high activity and content, EGCG seems to be the most effective antioxidant in all the components of green tea catechins. However, several researches showed that the tea catechin complex had a stronger effect than EGCG in the scavenging capacity of free-radical and anticarcinogenic activities (Shen et al., 1993
). This allows us to think that the constituents of tea catechin complex together have synergistic or additive effects on scavenging free-radical and cancer-preventive activity. Support for this activity was obtained from Suganumas study that (3H) EGCG incorporation into PC-9 cells was significantly enhanced by EC. Also, co-treatment with EGCG, EC, ECG, EC, EGC, and EC synergistically induced apoptosis of PC-9 cells and inhibited tumor necrosis factor-
release from BALB/ c-3T3 cells (Okabe et al., 1997
; Suganuma et al., 1999
).
Our previous research also demonstrated that various catechins in tea polyphenols constituted an antioxidant cycle, in accordance with the decreasing order of their first reductive potentials, and produced a coordinating, strengthening effect (Shen et al., 1993). As shown in Figures 6, 9, and 12
, the current data indicated that both EC and ECG significantly promoted the protective effect of EGCG. The mechanisms of action of ECG and EC are thought to be different because ECG did not stimulate EGCG incorporation into cells, whereas EC did (Suganuma et al., 1999
). Although the co-treatment with ECG and EGCG produced interesting results, the mechanisms of the action have not been well identified. Hashimoto et al.(1999)
found that ECG had the highest affinity for the lipid bilayer in membrane, followed by EGCG, EC, and EGC, with the partition coefficients of ECG in n-octanol/PBS being highest. Our former research suggested that the closer the first reductive potentials were, the more significant the coordinating and strengthening effects became (Shen et al., 1993
). Therefore, it is reasonable to deduce that the closer first reductive potential of EGCG and ECG, as well as their stronger affinity for lipid bilayer, allows them to easily enter the cell membrane and to show synergic effect. But the mechanism should be further investigated.
Recently, much attention has been paid to the prooxidant quality of natural products. It has been reported that, in the presence of the copper (II) ion under aerobic conditions, tea catechins induced DNA cleavage, accelerated the peroxidation of unsaturated fatty acid (Hayakawa et al., 1997), and killed Escherichia coli. (Kimura et al., 1998). These effects were apparently due to the prooxidant property of catechins. Our previous research also showed that both tea catechins complex and EGCG produced superoxide anion radical and semiquinone anion radicals in alkaline solution in vitro (Shen et al., 1992
). The results in this paper showed that even in the range of maximum nontoxic concentration, EGC demonstrated significant prooxidant signs, as shown in Figures 5 and 8
. These might be correlated with the toxicological effect of tea catechins. The investigation also placed catechins, under certain conditions, into radical-generating toxicological agents. Therefore, much consideration for safety should be required when tea catechins are used as therapeutic reagents or nutrition supplements.
Tea catechins are strong metal ion chelators because of the catechol structure (Guo et al., 1991; Kumamoto et al., 2001; Rice-Evans et al., 1997
). Though they have been shown to form stable complexes with Fe2+, Ca2+, Al3+, Mn2+, Cr3+, and Pb2+ (Guo et al., 1991; Kumamoto et al., 2001
), further investigation is needed as to whether tea catechins are capable of removing lead from the blood stream and target organs.
The present study was designed to elucidate whether tea catechins resulted in decreased lipid peroxidation in HepG2 cells treated by lead. The hypothesis was evidenced in tea catechin-treated HepG2 cells exposed to lead. Therefore it can be deduced that the increased cell viability in tea catechin-treated cells, along with improved lipid peroxidation levels, reflects the antioxidant action of tea catechins in lead-treated cells. Results from the study of cell membrane fluidity suggest that the beneficial effects of tea catechins on lipid peroxidation are related to its ability to protect cell membrane against damage by lead.
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ACKNOWLEDGMENTS |
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NOTES |
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