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Article

Supplementation of Jurkat T Cells with Green Tea Extract Decreases Oxidative Damage Due to Iron Treatment

Department of Food Science and Microbiology, Division of Human Nutrition, University of Milan, Milan, Italy

¹To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Cell supplementation and... Quantification of oxidative... RESULTS DISCUSSION REFERENCES

 
Regular tea consumption has been associated with a reduced risk of cancer. As demonstrated in vitro, green tea contains catechins with antioxidant properties. We evaluated the effect of the supplementation of the Jurkat T-cell line with green tea extract on oxidative damage. Cells grown in medium with or without green tea extract (10 mg/L) were treated with Fe²⁺ (100 µmol/L) as an oxidative stimulus for 2 h. Cell membrane lipid peroxidation was evaluated by fatty acids pattern analysis and malondialdehyde production in -linolenic acid–loaded cells. Furthermore, oxidative DNA damage (single strand breaks) was detected in cells by the Comet assay and quantified as relative tail moment (RTM). Supplementation with green tea extract significantly decreased malondialdehyde production (1.6 ± 0.3 vs. 0.6 ± 0.1 nmol/mg protein, P < 0.05) and DNA damage (0.32 ± 0.07 vs. 0.12 ± 0.04 RTM, P < 0.05) after Fe²⁺ oxidative treatment. In control cells, there was no effect on membrane distribution of (n-3) fatty acids due to Fe²⁺ treatment. Cell enrichment with -linolenic acid increased total membrane (n-3) fatty acids. However, the oxidative treatment did not modify the distribution of polyunsaturated fatty acids. It is likely that the observed protective effects can be attributed to epigallocatechin gallate, which is present mainly (670 g/kg) in green tea extract; however, we cannot exclude contributions by other catechins. These data support a protective effect of green tea against oxidative damage.

KEY WORDS: • catechins • Jurkat T cells • lipid peroxidation • DNA damage

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Cell supplementation and... Quantification of oxidative... RESULTS DISCUSSION REFERENCES

 
Several epidemiologic studies have suggested an association between green tea consumption and a reduced risk of different kinds of human cancer (Katiyar and Mukhatar 1996 , Yang and Wang 1993 ). In addition, the antioxidant activity of tea flavonoids has been related to a protective effect on human health by the prevention of degenerative diseases such as coronary heart disease (Hertog et al. 1993 , Keli et al. 1996 , Kono et al. 1992 , Weisburger 1999 ). The main constituents of green tea flavonoids are catechins, among which epigallocatechin gallate (EGCg)² and epigallocatechin (EGC) are the most common (Salah et al. 1995 ). They have potent antioxidant properties, including the scavenging of oxygen radicals and lipid radicals (Husain et al. 1987 , Torel et al. 1986 ). Many in vitro models have been used to test flavonoid antioxidant activity. Human LDL oxidation, measured by conjugate dienes, was delayed by phenolic agents; the concentration of EGCg giving 50% inhibition of LDL oxidation was 71 µmol/L (Wang and Goodman 1999 ). An LDL model was also used by Salah et al. (1995) who demonstrated a higher antioxidant potential evaluated as trolox equivalent antioxidant activity for EGCg with respect to other polyphenols. Furthermore, epicatechin (EC) and epicatechin gallate (ECg) (10 µmol/L) showed a protective effect against lipid peroxidation in phospholipid bilayers (Terao et al. 1994). On the other hand, an increase of total antioxidant plasma activity was detected in humans ~30 min after a single intake of 300 mL green tea (20 g/L tea leaves) (Serafini et al. 1996 ).

However, the absorption and mechanism of action of these compounds in cells such as lymphocytes, which are primary targets of oxidative damage, have not been yet evaluated. Cell culture has been used mainly for studying chemopreventive action of catechins at relatively high concentrations (Chen et al. 1998 , Lea et al. 1993 , Ramanathan et al. 1992 ), whereas it seems that the antioxidant function occurs at lower levels.

The aim of this work was to evaluate the efficiency of catechins from green tea extract in preventing lipid peroxidation and DNA damage in the Jurkat cell line when subjected to oxidative stress. T lymphocytes have been widely used as target cells to investigate cell response to oxidative stress (Duthie et al. 1996 , Riso et al. 1999 ). We chose the Jurkat cell line, in particular, because their membrane markers make them resemble normal lymphocytes (Konicova et al. 1992 ). For an oxidative stimulus, we chose iron because it is normally present in cells and it has been used by other investigators (Burns and Wagner 1991 , Kelley et al. 1995 ) as a model compound with which to investigate the effects of oxidative stress in cell culture. Treatment with Fe²⁺ produces hydroxyl radicals via a Fenton-type reaction that is able to cause oxidative damage to cells. In particular, we evaluated lipid peroxidation by analysis of membrane fatty acid patterns, malondialdheyde (MDA) production and single strand breaks in DNA by Comet assay.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Cell supplementation and... Quantification of oxidative... RESULTS DISCUSSION REFERENCES

 
Cells and reagents.

The Jurkat human leukemia cell line was cultured in RPMI 1640 containing 100 mL/L newborn calf serum, 2 mmol/L of L-glutamine, 1 x 10⁵ IU/L penicillin, 100 mg/L of streptomycin at 37°C in a humidified atmosphere of 5% carbon dioxide/95% air. Cells were grown in 275 mL flasks and medium was changed every 48 h, adjusting the cell number to 3.5 x 10⁸ cells/L after hemocytometer counts. Viability of cells was assessed by the trypan blue exclusion assay (Cook and Mitchell 1989 ) and by the 3,–4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) assay (Marks et al. 1992 ).

Reagents were purchased from Sigma Chemical (St. Louis, MO) and Merck (Merck KGaA, Darmstad, Germany), and standards of catechins from Extrasynthese (Genay, France). The green tea extract, Greenselect, was obtained from Indena (Indena SpA, Milan, Italy) and analyzed by HPLC for its catechin concentration.

Catechins analysis.

HPLC analysis was performed using a model 510 pump (Waters, Milford, MA) equipped with a Rheodyne injector coupled with an electrochemical Coulochem II detector (ESA, Chelmsford, MA). The column was a Symmetry C18, 5 µm (250 x 4.6 mm, i.d.) from Waters. Catechins and gallic acid were eluted using a linear gradient from 0 to 25% acetonitrile in 30 mmol/L NaH₂PO₄ buffer (pH 3) in 30 min. The flow rate was 1.3 mL/min. The parameters for the electrochemical detector were as follows: guard cell, -350 mV; Cell 1, -100 mV; Cell 2, +350 mV; sensitivity, 50 nA. Green tea catechins (EGCg, ECg, EGC, EC and gallic acid) were dissolved in methanol (1 g/L) and stored at 0°C. Aliquots of standard solutions in the range from 5 to 50 µg/L were injected into the HPLC apparatus. The limit of detection, calculated as the concentration producing a peak height three times the baseline noise, was 1 µg/L.

Study design.

We studied the efficiency of green tea supplementation in preventing lipid peroxidation and DNA damage in cells. Fatty acid profiles and MDA production were analyzed to detect lipid peroxidation. Fatty acid profiles were investigated in control cells and -linolenic acid (LNA)-loaded cells that were or were not subjected to the oxidative treatment. Malondialdheyde, a naturally occurring product of the oxidation of PUFA, was detected in LNA-loaded cells that were or were not supplemented with green tea extract and subjected to the oxidative treatment. The enrichment with LNA was necessary to increase the substrate for oxidation. Control and treated cells that were or were not supplemented with green tea extract were analyzed for DNA damage.

	Cell supplementation and treatments

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LNA enrichment.

This was achieved by growing cells for 72 h in media supplemented with LNA at 32 µmol/L (Burns and Wagner 1991 ). Enriched cells were tested for viability. LNA was dissolved in absolute ethanol, neutralized with NaOH and dried under nitrogen. The sodium linolenate was then dissolved with 2 mL distilled water at 30°C; this solution was added to the heat-inactivated (56°C for 30 min) newborn calf serum and stirred (Mooney and Lane 1981 ).

Green tea extract supplementation.

Greenselect was dissolved in medium and added to the cell culture to a final concentration of 10 mg/L and maintained for 24 h at 37°C. The suspension was then washed twice with PBS before the oxidative treatment, which was performed in PBS by adding 100 µmol/L Fe²⁺ (as FeSO₄) for 2 h. Cells were then centrifuged for further analysis (400 x g, 10 min).

	Quantification of oxidative damage

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Fatty acid analysis.

The modification of the lipid profile was assessed by gas-chromatographic (GC) analysis of cell membrane phospholipids. Total lipids were extracted by the method of Folch et al. (1957). For phospholipid isolation, the extract was eluted into a silica cartridge (Sep-Pack plus silica, Waters) and then collected with methanol/toluene (4:1). Methylation was obtained with acetyl chloride at 100°C for 1 h (Liebich et al. 1991 ). After centrifugation at 800 x g for 10 min, the supernatants were stored at -20°C. Before analysis, the samples were dried under nitrogen and dissolved with hexane. Methyl esters were separated by GC (Varian GC 3400, Mulgrave, Victoria, Australia) using an Omegawax 320 column (30 m x 0.32 mm i.d., 0.25 µm film) from Supelco (Bellafonte, PA). Peak areas were quantified and identified by comparison of retention times with those of standards obtained from Sigma Chemical.

Determination of MDA.

A suspension of ~1 x 10⁷ cells in 25 mL of medium was used for MDA determination. After the procedure (LNA enrichment, with or without green tea supplementation and with oxidation), cells were centrifuged and resuspended with 0.5 mL of distilled water and sonicated at 40% for 1 cycle of 5 s; 50 µL were taken for protein analysis. Protein concentrations were assessed by the method of Lowry et al. (1951).

To the remaining sample, 0.45 mL of acetonitrile was added, mixed and centrifuged for 5 min at 3300 x g. The supernatant was immediately analyzed by a method modified from Esterbauer et al. (1984) . The HPLC system consisted of a model 501 pump (Waters) and a UV/VIS detector (model 486, Waters). A Spherisorb-NH₂ 5-µm (250 x 4.6 mm) column (Alltech, Milan, Italy) was eluted isocratically at 1.5 mL/min with a 70:30 mixture of acetonitrile/0.03 mol/L Trizma buffer, pH 7.4, and detected at 267 nm. The standard of MDA was obtained by acid hydrolysis of 1,1,3,3-tetramethoxypropane by the method of Esterbauer et al. (1984) . A calibration curve (3–21 µmol/L) was used for MDA quantification, and results were expressed as nmol MDA/mg protein. A photodyode array detector supported by the Millenium 2010 Chromatography Manager computing system (Waters) was used to confirm peak identification, registering the spectra in the range between 200 and 400 nm.

Determination of DNA damage by Comet assay.

The assay was applied as previously described (Riso et al. 1999 ) and used to evaluate DNA damage in control and supplemented Jurkat cells that were or were not subjected to the oxidative treatment. For the analysis, 100 µL of cell suspensions (2.5 x 10⁶ cells in 5 mL of medium) was centrifuged at 5000 x g for a few seconds at room temperature and the pellet resuspended in 50 µL of PBS.

Briefly, cells embedded in agarose were lysed and then subjected to electrophoresis under alkaline conditions; after neutralization, they were stained with ethidium bromide. With this assay, cells with increased DNA damage displayed increased DNA migration from the nucleus towards the anode (Comet).

Cell images for each slide (n = 50) were electronically captured and analyzed for fluorescence intensity with a Comet analysis program supported by the image processing environment Visilog 4 (Noesis, Orsay, Cedex, France). To quantify the DNA damage, the tail and head moments were evaluated as follows: tail/head moment = sum of the intensity of each pixel in the tail/head multiplied by its distance from the center. From these two parameters, the relative tail moment (RTM) was calculated as follows: RTM = tail moment/(head moment + tail moment). RTM of treated and control cells were evaluated.

Statistical analyses.

Statistical analyses were performed on a personal computer using the Statistca Software (Stat Soft, Tulsa, OK). A repeated-measures ANOVA design with treatment as independent factor was used to investigate the effect of the different experimental conditions on DNA damage and fatty acids pattern. For the DNA damage, the treatments were as follows: control, control + green tea (GT), control + oxidation (OX), GT + OX; for fatty acids, the treatment patterns were as follows: control, control + OX, LNA-enriched cells, LNA-enriched cells + OX. Differences between means were further evaluated by the Least Significant Difference test. The effect of green tea supplementation on MDA production was analyzed by t test. P-values < 0.05 were considered significant.

	RESULTS

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Characterization of green tea extract.

The composition of green tea extract and final catechin concentration in medium are reported in Table 1 . The total catechin concentration in the extract was 786 g/kg. The main component present was EGCg at a final concentration in medium of ~15 µmol/L.

Cell viability and morphology were not significantly affected by the different treatments.

Fatty acid modification.

GC analysis of enriched medium showed a consistent increase in LNA level compared with the control medium (data non shown). After LNA supplementation, the fatty acid composition of the membrane of Jurkat cells showed an enrichment in LNA (1.68 g/100 g fatty acids) compared with control cells (not detectable) (Table 2 ). Moreover, there was also an enrichment in the elongation and desaturation products of LNA such as 18:4(n-3), 20:4(n-3), 20:5(n-3) and 22:5(n-3) (0.6, 1.78, 2.76 and 3.63 g/100 g, respectively). The total (n-3) level was greater in LNA-enriched cells than in control cells.

View this table: [in this window] [in a new window]   Table 2. Fatty acid pattern in control and -linolenic acid (LNA)-enriched cells with or without oxidative (OX) treatment1

The proportions of (n-3) and (n-6) were unaffected by oxidative treatment in both control and LNA-enriched cells; thus peroxidative damage was not assessed by this method (Table 2) .

MDA production.

MDA analysis was performed in LNA-enriched cells subjected to the oxidative treatment with or without green tea supplementation. MDA concentration was not detected in PBS after oxidation, excluding MDA released from cells. After oxidative treatment, MDA concentration was significantly lower (P < 0.05) in cells supplemented with green tea extract (0.6 ± 0.1 nmol/mg protein; mean ± SEM, n = 3) than in cells without green tea supplementation (1.6 ± 0.3 nmol/mg protein , n = 3).

DNA damage.

Treatment of control cells with Fe²⁺ caused more DNA damage than in untreated control cells (Fig. 1 , P < 0.01). Supplementation of Jurkat cells with 10 mg/L of green tea extract did not increase DNA single strand breaks. On the contrary, oxidized cells previously supplemented with green tea extract resisted DNA damage (P < 0.05).

View larger version (25K): [in this window] [in a new window]   Figure 1. DNA damage evaluated by the Comet assay and expressed as relative tail moment (RTM) in control and oxidized (OX) cells (Fe2+, 100 µmol/L for 2 h), supplemented or not with green tea extract (GT; 10 mg/L). Values are means ± SD, n = 3. Means with unlike letters differ, P < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Cell supplementation and... Quantification of oxidative... RESULTS DISCUSSION REFERENCES

It has been suggested that most of the lipid peroxidation observed in vivo is metal ion dependent, often involving iron and sometimes copper. Iron ions participate in Fenton chemistry, generating hydroxyl radicals that are particularly reactive with lipid. Thus, the most biologically relevant evaluation of ability to inhibit lipid peroxidation can be made by testing an antioxidant against metal ion–stimulated lipid peroxidation in biological membranes (Halliwell 1995 ). It has been reported that 20 µmol/L is approximately the physiologic concentration of iron bound to transferrin in human blood. However, Burns and Wagner (1991) , using 20 µmol/L Fe²⁺ in L1210 leukemia cells, did not find any modification of the proportion of (n-3) and total unsaturated fatty acids. In a subsequent study, a higher concentration of Fe²⁺ (100 µmol/L) was used with the same cellular line, and this concentration induced oxidative stress (Kelley et al. 1995 ). In this study, the fatty acid pattern of cell membranes in control and LNA-loaded cells was not affected by treatment with 100 µmol/L Fe²⁺ for 2 h. Consequently, the analysis of cells subjected to the oxidative treatment after green tea extract supplementation was not performed. These results suggest that the modification of membrane fatty acid composition does not seem to be a useful variable for the study of peroxidation, hence its prevention by antioxidants. In this study, another marker of lipid peroxidation, MDA production, was measured by HPLC in cells grown in LNA-enriched medium, thus avoiding the nonspecificity of the thiobarbituric acid test (Kishida et al. 1993 ). The enrichment with LNA was necessary to increase the substrate for oxidative treatment and make MDA detectable by HPLC. Malondialdehyde production due to Fe²⁺ treatment was significantly lower in LNA-loaded cells supplemented with green tea extract than in unsupplemented LNA-loaded cells. It has been hypothesized that polyphenols act as antioxidants by chelating metal ions or acting as hydrogen-donating radical scavengers. As already reported by others, catechins might be localized close to the membrane surface, possibly bound to it, to scavenge aqueous radicals and thus prevent lipid peroxidation (Salah et al. 1995 ).

Oxidative treatment can also induce DNA damage; hydroxyl radicals are thought to be responsible for most of the injuries that take the form of strand breaks and oxidized bases. It has been suggested that substantial oxidative DNA damage in vivo contributes to the etiology of cancer. Consequently, the quantification of DNA damage is a useful biomarker of the oxidative status and the antioxidant defense system of the cell (Riso et al. 1999 ). We evaluated DNA damage using the Comet assay, a sensitive procedure for detecting DNA single strand breaks in any population of eukaryotic cells that can be obtained in a suspension of single cells.

Supplementation with 10 mg/L (15 µmol/L EGCg) green tea extract did not result in a modification in basal DNA damage with respect to control cells. In contrast to our study, Duthie et al. (1997) found that other flavonoids such as quercetin, myricetin and silymarin induced strand breaks in DNA in a dose-dependent manner (>100 µmol/L), as evaluated by the Comet assay.

In this study, the treatment of cells with Fe²⁺ caused significant DNA damage in control cells. On the contrary, cells previously supplemented with the green tea extract were protected from oxidative stress as demonstrated by the low DNA damage registered, which was not different from that of control cells.

This finding is particularly interesting because it suggests that catechins can prevent DNA damage independently from lipid peroxidation. They could act simply as scavengers of free radicals at the membrane level or may be responsible for an improvement of the whole antioxidant defense system of the cell.

To our knowledge, no other papers have reported the effect of the supplementation with catechins on cell resistance to oxidative DNA damage. Recently, Noroozi et al. (1998) , used the Comet assay to assess the antioxidant potencies of several dietary flavonoids such as quercetin, luteolin and rutin on oxygen radical–generated DNA damage from hydrogen peroxide (100 µmol/L) in human lymphocytes supplemented ex vivo. The authors calculated that 47 µmol/L of quercetin was necessary to obtain a 50% reduction in human lymphocyte DNA damage. They incubated cells with quercetin (from 7.6 to 279.4 µmol/L) for only 30 min and found a dose-response inverse relationship between oxidative DNA damage and flavonoid concentration.

Catechins are flavanols, and several in vitro studies (Salah et al. 1995 , Terao et al. 1994, Wang et al. 1999 ) reported a relative antioxidant potential of EGCg comparable to that of quercetin. Furthermore, Vinson et al. (1995) , using an in vitro lipoprotein oxidation model, found that catechins were the most powerful antioxidants of the flavonoids (including quercetin).

In this study, 24-h incubation of Jurkat cells in ~15 µmol/L of EGCg was enough to significantly reduce DNA damage. However, green tea extracts contain minor quantities of other catechins whose antioxidant properties could have contributed to the effects observed.

Moreover, under our conditions, a quantity of green tea extract >10 mg/L (~15 µmol/L EGCg) resulted in decreased cell viability. Chen et al. (1998) demonstrated growth inhibition in different transformed cell lines after supplementation with 40 µmol/L EGCg and suggested that cancer cells are more susceptible to the action of EGCg than are normal counterparts cells. Duthie et al. (1997) found growth inhibition of human lymphocytes after only 18 h of supplementation with 10 µmol/L quercetin.

Data in the literature do not agree concerning the effects of different concentrations of flavonoids on cell growth (Lea et al. 1993 , Ramanathan et al. 1992 , Valcic et al. 1996 ). The effects of this class of compound on viability and antioxidant capacity are closely related to the experimental conditions such as the type of cell considered, the flavonoid concentrations and the duration of treatment. Further studies are required to verify the antioxidant action of these compounds consistent with cell viability.

The results we obtained support the protective effect of catechins present in green tea extract (10 mg/L) against oxidative damage in Jurkat cells. In an in vivo study, with an intake of 400 mg EGCg as a tablet (Greenselect), Pietta et al. (1998) found a 2 µmol/L increase in EGCg plasma concentration and an increase in the plasma total radical antioxidant parameter compared with plasma obtained from control subjects. In our cellular model, a concentration of EGCg about eight times higher (15 µmol/L) improved cell protection from membrane lipid peroxidation and DNA damage. The in vivo counterpart of these findings seems to support the rationale for nutritional advice directed to increase green tea consumption to prevent cell oxidative damage and related diseases. The effectiveness of low concentrations of green tea extract (comparable to that achievable after green tea infusion intake) should be further evaluated in cell culture and in vivo studies.

	ACKNOWLEDGMENTS

 
We are grateful to INDENA SpA for the gift of the green tea extract sample. Claudio Gardana is acknowledged for catechin analysis and Stefano Ravasenghi for MDA analysis.

	FOOTNOTES

 
² Abbreviation used: EC, epicatechin; ECg epicatechin gallate; EGC, epigallocatechin; EGCg, epigallocatechin gallate; GC, gas chromatography; LNA, -linolenic acid; MDA, malondialdehyde; MTT, 3,–4,5-dimethylthiazol-2,5-diphenyl tetrazolium bromide; RTM, relative tail moment.

Manuscript received May 11, 1999. Initial review completed June 18, 1999. Revision accepted August 23, 1999.

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