U937 CELLS AS A MODEL TO STUDY THE EFFECT OF PHYTOCHEMICALS ON SUPEROXIDE ANION PRODUCTION

NutritionResearch,Vol.17,No.7, pp. 1091-1103,1997 Copyright@ 1997ElsevierScienceInc.

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U937 CELLS AS A MODEL TO STUDYTHE EFFECT OF PHYTOCHEMICALSON SUPEROXIDEANION PRODUCTION G. R. FroemrningMSC.,Ph.D. and N. M. O’Brien MSC.,Ph.D.* Department of Nutrition, University College, Cork, Republic of Ireland

ABSTRACT The ability of phytochemicals to suppress production of superoxide anion (02””) in a cell culture model was assessed. Differentiated U937 cells and human polymorph nuclear leukocytes (PMN) were stimulated with increasing concentrations of phorbol myristate acetate (PMA) and 02-”release was measured by cytochromec reduction. The overall trend in 02-”production in response to PMA stimulation was similar in both U937 cells and PMN. The effect of the phytochemicals, chlorogenic acid, caffeic acid, curcumin, quercetin, (-)epicatechin and glycyrrhizic acid (50, 100 and 500 nM) on 02-”formation from PMA stimulated U937 cells was examined. All phytochemicals suppressed 02-”release from U937 cells with chlorogenic acid, (-)-epicatechin and glycyrrhizic acid showing dose dependent effects. In conclusion, U937 cells are a usefid model to screen phytochemicals for their ability to suppress 02-”formation. @1991 ElsevierScimcelnc. Key words: U937 cells, PMA, Superoxideanion, Phytochemicals

INTRODUCTION Superoxide anion (02”’) in combination with other reactive oxygen species (ROS) such as H202 and OH” are important mediators of inflammatory response, ischemia-reperfhsioninjury and atherosclerosis (1, 2). ROS generated by activated macrophages during prolonged inflammation may cause irreversible oxidative damage to neighboring cells. ROS have also been shown to damage DNA and other macromolecules, to be mutagenic and to play a role in the carcinogenic process (3, 4, 5, 6, 7). Tumor promoters, e.g. phorbol myristate acetate (PMA), are known to stimulate the production of 02-’by activation of the NADPH-oxidase,a membrane-bound enzyme, in phagocytes. Protein kinase C (PKC) is a mediator of PMA induced NADPH-oxidase activation (8, 9). Studies on white blood cells, HL 60 cells (10), human tumor cells (11) and HeLa-cells (12) indicated production of 02-”and H202 in response to PMA stimulation. Furthermore, PMA caused oxidized DNA bases formation in HeLa-cells (12) and increased xanthine-oxidase activity in NIH 3T3 cells (13). Many turnor promoters provoke a rapid decrease in cellular antioxidant defenses, including superoxide dismutase, catalase, and glutathioneperoxidase activities (14, 15). In this work, we investigated several naturally occurring phytochemicals as potential inhibitors of 02-’production in a PMA-stimulated phagocytic cell line. Phytochemicals are nonnutrient components found in fruits, vegetables, spices, herbs and beverages like wine and tea. The *Corresponding author:Dr.NoraO’Brien,DepartmentofNutritionuniversityCollegeCork,RepublicofIreland, Phone+353-21-90-2884, Fax+353-21-270244. e[[email protected] 1091

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majority of these compounds are phenolic acids, flavonoids and catechins. Humans ingest up to one gram of these compounds per day. Caffeic acid and chlorogenic acid are widely distributed in the plant kingdom and are common constituents of beverages, fruits and vegetables consumed by man (16, 17). Both acids have been shown to have anticarcinogenicproperties (18, 19) and to be active inhibitors of PMA-induced tumor promotion in mouse skin and other animal models (20). They also inhibit the metabolism of arachidonic acid (21). Curcumin, contained in the rhizome of the plant Curcuma Lorzga .Lirm, is the major yellow pigment in curry, turmeric and mustard and is widely used as a coloring agent in foods. Curcuminhas been used widely in India and Indonesia for the treatment of inflammation. It is a potent inhibitor of PMA-induced tumor promotion, arachidonic acid metabolism in mouse skin and epidermal lipoxygenase and cyclooxygenase activity (22, 23). Quercetin is found in tea, coffee, cereal grains, red wine and fruits and vegetables. Some foodstuffs contain significant amounts of quercetin e.g. onion (280 -480 mg/kg) or kale (110 mg/kg). As a flavonoid, it is a strong inhibitor of several enzyme systems such as Iipoxygenaseand cyclooxygenase. It also inactivates protein kinase C (24), which is involved in the activation of NADPH-oxidase. Quercetin is an inhibitor of tumor development in several experimental animal models (25, 26, 27). (-)- Epicatechin is a constituent of green tea. Green tea has a wide range of biological activities and has been shown to inhibit tumor promotion in vivo and vitro (28) and to enhance the immune system (20). Glycyrrhizic acid is the main principle of licorice, derived from the rhizome of Glycyrrhiza plants. Glycyrrhizic acid has antiinflammatory and antiallergic effects (29).

Previous studies examining the effect of phytochemicals on Oz” formation by phagocytic cells have used mainly primary cultures of human neutrophils (e.g. polymorphonuclearleukocytes, PMNs) as a model (30, 31, 32). Use of this model depends on the availability of appropriate human volunteers and trained phlebotomists, in addition, these cells experience a reduced ability to produce 02-’with time. U937 cells, used in this study, are an immature monocyte-like cell-line. They share many characteristics with peripheral blood monocytes and, when induced to differentiate along the monocyte-microphage lineage, they acquire the ability to produce 02-”. Therefore, we studied 02-” production by both PMA-stimulated U937 cells and PMN. The purpose of using the two cell types was to demonstrate that differentiated U937 cells can, like PMNs, be stimulated to produce 02-”. While use of cell lines such as U937 cells has limitations, consistency of effects seen in a variety of in vitro models strengthens the findings. In addition, the U937 cell line has been widely used as a model for studying human monocyte maturation and function (33, 34, 35, 36, 37, 38). The scavenging effects of caffeic acid, chlorogenicacid, curcumin, quercetin, (-)-epicatechin and glycyrrhizic acid on PMA induced 02” generation in U937 was investigated. The 02”’ scavenging effects of these phytochemicals were also evaluated in a cell-free model using xanthineixanthine-oxidase.

MATERIALSAND METHODS

Reagents. Phorbol 12-myristate 13 acetate (PMA), calcium ionophore A 23187, N-formyl methionyl leucyl phenylalanine (FMLP), xanthine-oxidase (Grade III from Buttermilk), xanthine (2,6-dihydroxypurine),cytochrome c (from horse heart), superoxide dismutase (SOD from bovine erythrocytes) and MEM non-essential amino acid solution were obtained from Sigma (Poole, Dorset, UK). Lymphoprep TM(density 1.OTT+ o.001 g/ml) was purchased from Nycomed pharma AS (Oslo, Norway) and the RPMI-1640 medium, the fetal calf serum and all antibiotics from Gibco

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(Paisley, Scotland). Caffeic acid (3,4 dihydroxycinnamic acid) was purchased from Aldrich (Gillingham, Dorset, UK), quercetin (3, 3’,4’,5,6-pentahydroxyflavone), .(.) epicatechin, glycyrrhizic acid (3-O-[2-O-fi-D-glucopyr~wonosyl-~-D-glucopyrmWonosyl]-1813-glycyrrhetinic acid ammonium salt) from Fluka (Gillingham, Dorset, UK) and chlorogenic acid (1,3,4,5 tetrahydroxycyclohexane-carboxylicacid) and curcurnin (1,7-bis[4-hydroxy-3-methoxyphenyl].1,6heptadiene-3,5-dione)from Sigma (Poole, Dorset, UK). Isolation of PMN. Blood samples were taken from informed healthy adult donors after venipuncture and collected in heparinized tubes. PMN were separated by a modification of the method of Limasset et al. (30). Briefly, 10 ml of diluted blood was layered onto 20 ml of lymphoprebTMand centrifuged at 2500 rpm for 30 min after which the lymphocyte and monocyte layers were removed. Neutrophils were then separated from erythrocytesby dextran sedimentation. The supernatant containing the neutrophils was removed and centrifuged at 2500 rpm for 10 min. The pellet containing neutrophils was incubated with lyse buffer for 10 min to lyse any remaining erythrocytes. PMN were collected after further centrifitgationand washed 3 times with 0.9 0/0 NaCl solution. The cell viability was at least 98 0/0 as assessed by tryphan blue exclusion. The cell density was adjusted to 1 x 106cells/ml and the cells were plated into 24-well multiwell plates. U937 cell line. U937 cells were a gift from the Microbiology Department, University College, Cork. The cells were grown in RPMI-1640 media supplemented with glutamine (2 mM), nonessential amino acids (5 ‘Yo), penicillin/streptomycin (100 units/ml), fimgizone (5 units/ml) and gentamicin (2 mg/ml) to which 10 ‘%0 fetal calf serum was added. U937 cells were cultured in a humidified incubator (Forma Scientific) at 37 ‘C and 5 VO C02. Cell viability was determined by trypan blue exclusion in a hemocytometerchamber. Differentiation of U937 cells. To obtain differentiated cells, U937 cells (5 x 105cells/ml) were grown in the presence of 80 nM of PMA for 3 days. At the end of the 3 day period the PMA was removed and cells were washed 2 times with HBSS (composition in mM: NaCl 137.25, KC15.56, NazHP04 0.34, KHZP040.46, CaC12x 2 HZO1.27,MgSOqx7H200.86, glucose 5.0, Hepes 25.0, pH 7.4). PMA was made up as a stock solution of 1.62mM in DMSO and kept frozen at -20”C. Measurement of superoxide anion release. Superoxide anion release was estimated using a modification of superoxide dismutase-inhibitable reduction of cytochrome c assay (38). Briefly, cells were washed 2 times with HBSS and then incubated with HBSS containing 52 PM cytochrome c and various concentrations of PMA. For each parameter assessed, half of the wells were incubated with 300 units of SOD/ml and half without. The final volume of the reaction mixture was 1 ml. After stimulation the reaction was stopped by placing the dishes on ice. The samples were centrifuged at 14000 rpm for 4 min and absorbance read in a Shimadzu spectrophotometer (UV-120-02) at 550 nm. The amount of 02-”released was determined by dividing the average difference in absorbance in samples cultured with and without SOD by extinction coef%cientfor the reduced cytochrome c (e=21.1 mmol-l cm-l) (39). Measurement of 02-’derived tlom xanthinelxanthine-oxidasesystem was done using a modification of Chatham et al. (40). 02-’production was induced by mixing 30 PM xanthine, 0.04 units xanthine oxidase and 52 I.LMcytochrome c in HBSS. The reaction mixture was left at room temperature for 10 min before reading spectrophotometricallyat 550 nm. The amounts of superoxide anion was calculated using the extinction coefficient for the reduced cytochromec, as above. Inhibition of superoxide anion production by phytochemicals. Phytochemicals were prepared as a stock solution in ethanol (10 mM) and stored at -20° C. Cells were preincubated with 50, 100

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or 500 nM of the phytochemical for 3h before the superoxide anion release assay was carried out. Control cells were treated with PMA only. Oz-’production by control cells was designated as 100 ‘X.. In case of the cell-free Oz-’system, the phytochemicals were incubated together with xanthine, xanthine-oxidase and cytochrome c. To rule out a possible interference by the phytochemicals on the 02-”release assay, a control group was included. The control contained the phytochemical, xanthine and cytochrome c. The readings obtained for the control were subtracted from the system containing xanthine-oxidase. 02-”produced by xanthinelxanthine-oxidasewithout the presence of the phytochemicals was designated as 100 ?40. Statistical analysis. Results are presented as mean values and standard errors of the means. Data were analysed by one-way analysis of variance (ANOVA) followed by least significant difference (LSD). The level of statistical significancewas taken as p <0.05.

RESULTS

Comparison of superoxide anion release by differentiatedU937 cells and PMN. When exposed to 80 nM PMA for 3 days, U937 cells differentiatedfrom a monocyte-likecell to a microphage like cell. They became adherent (} 80 0/0), formed clumps and expressed the phenotypic properties of macrophages. Cell viability, assessed by trypan blue exclusion was higher than 90 ‘%. in both undifferentiated and differentiated cells. When fin-therstimulated with PMA, the differentiated U937 cells (5 x 105cells/ml) released 02-”. The amount of 02-’was dependent on incubation time (30 and 60 rein) and PMA concentration (16 -324 nM). After exposure for 30 min using low concentrations of PMA (O- 81 nM), there was an increase in 02””production which plateaued afler 81 nM PMA (FIG. la). After 60 min of stimulation with PMA, the amount of 02-”released was higher at all concentrations tested, reaching a maximum (2.4 + 0.5 nmol Oz-’)at 81 nM PMA. PMA, at the concentrations and exposure times used, had no damaging effect on U937 cells, as assessed by LDH-leakage and neutral red uptake (data not shown). PMN were also stimulated for 30 and 60 min with increasing concentrations of PMA. After 30 min incubation time the highest release of 02-’was 6.9 + 1.0 nmol for 41 nM PMA (FIG. lb). With 60 min of exposure to PMA the higher release of 02-”(9.1 + 1.3 nmol) occurred at 81 nM PMA but fell to lower level of release (5.4 + 1.3 nmol) with increasing concentrations of PMA (FIG. lb). The overall trend of Oz-”release observed in PMN was similar to differentiated U937 cells. At the concentrations and times used, PMA had no damaging effect on PMN as assessed by LDH-leakageand neutral red uptake (data not shown).

Comparison of different stimuli on superoxide anion release in both differentiated U937 cells and PMN. The effect of different stimulators including PMA, FMLP and Ca-ionophoreA23187 on 02-’release in both differentiated U937 cells and PMN is shown in FIG. 2. The concentrations of PMA, FMLP and Ca-ionophore A23187 that induced the highest 02-’production in both cell types as determined from dose-responsecurves (data not shown) are presented in FIG. 2. U937 cells gave the strongest response, in terms of 02-’production, after stimulation with PMA (2.4 + 0.4 nmol) and FMLP (2.5 + 0.8 nmol) followed by Ca-ionophoreA 23187 (1.3 + 0.7 nmol). The PMN responded strongly to PMA stimulation (6.1 + 0.6 nmol). The amounts of 02-”released from PMN, following incubation with FMLP (5 ILM)and Ca-ionophoreA 23187 (0.5 PM) were 1.6 + 0.4 nmol Oz-’and 2.8 + 0.2 nmol 02-”respectively. FMLP was a weak stimulator of 02-. production in PMN but a good stimulator in differentiated U937 cells (2.5 + 0.8 nmol ). The stimulators PMA and FMLP

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induced similar amounts of Oz-”production in differentiated U937 cells. Based on these results we selected PMA as a stimulant in all subsequentexperiments. Effect of phytochemicals on superoxide anion release from PMA stimulated U937 cells. U937 cells werepreincubatedwith 50, 100and 500 nM of either chlorogenicacid, caffeic acid, curcumin, (-)-epicatechin, quercetin or glycy~hizic acid for 3 h. Unstimulated cells represented base level production of Oz-’by differentiated U937 cells (FIG. 3). For a better comparison of results, the Oz-’ release from PMA stimulated cells was set at 100 O/O. The concentration of PMA used in these experiments was 162 nM. There was no significant difference in 02-’release from cells treated with 81 and 162 nM PMA. There was a significant increase in 02- production in cells stimulated with PMA compared to unstimulated cells. This was consistently seen in all experiments (FIG. 3). At all concentrations of chlorogenic acid examined there was a significant reduction in the amount of 02-”released from the PMA stimulated cells (FIG. 3 ). The inhibition seemed to be dose dependent but did not reduce 02-”production to the level seen in unstimulated differentiated U937 cells. Caffeic acid reduced significantly 02-”production at all concentrationstested. (-)-Epicatechin, like chlorogenic acid, inhibited significantly 02”’release from stimulated cells in a dose-dependent manner. Quercetin, curcumin and glycyrrhizicacid at all concentrationstested reduced significantly the amount of 02-”released from stimulated cells. Effect of phytochemicals on xanthinelxanthine-oxidase superoxide anion production. Chlorogenicacidhadlittle effect on 02””productionin the xanthinelxanthine-oxidaseassay (FIG. 4). While there was a decreasing trend in 02-”release with increasing concentrations of chlorogenic acid, only at the higher concentration tested (500 nM) was there a significant difference between treated and control. Caffeic acid was a weak inhibitor of xanthinelxanthine-oxidaseproduced 02-”at the lower concentrations tested (50 and 100 nM). A concentration of 500 nM caffeic acid f~her decreased 02-”production by xanthinefxanthine-oxidase. Similarly, quercetin had little effect in reducing Oz-”production by xanthine-oxidaseand again, a significantdifference between treated and control was seen only at the higher concentrations tested (500 nM). In contrast (-)- epicatechin, curcumin and glycyrrhizic all inhibited significantly Oz- production. Glycyrrhizic acid decreased Oz-’production in a dose-dependentmanner.

DISCUSSION

U937 cells are a human monocyte/microphage cell line derived from a histiocytic lymphoma and share many characteristics of human monocytes (41). U937 cells are very susceptible to morphological and biochemical differentiation following exposure to agents as diverse as phorbol esters, dimethyl sulfoxide, cyclic nucleotides or vitamin D3 and others (33). When induced to differentiate along the monocyte-microphage lineage, U937 cells undergo morphological and functional changes, acquiring the ability to respond to chemotactic agents, produce 02-”,release lysomal enzymes, and perform antibody-dependentcytolysis (34, 35). Binding of PMA to its special cell-surface receptor on neutrophil and other phagocyte membranes leads to assembly of the NADPH-oxidase complex with resultant extracellular secretion of superoxide (42). Evidence indicates that the NADPH-oxidase may be responsible for 02-’generation in U937 cells (35). In this study we demonstratedthat U937 cells are capable of behaving like other phagocytes in terms of 02””production. They released 02-”upon stimulation with PMA, FMLP and Ca-

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ionophore A 23187 (FIG. 2). Routine use of primary cultures of PMN necessitates obtaining blood from appropriate human volunteers. Therefore, the use of cell lines is advantageous, because the supply of cells is more constant. Using U937 cells, we found a good and reproducible activation of the U937 cells by various stimuli (FIG. 2a). Superoxide radicals are mainly generated from several different enzymatic systems. The NADPH-oxidase system in U937 cells (35) and the xanthine-oxidasesystem (42) were examined in this study. The phytochemicals chlorogenic acid, (-)-epicatechin and glycyrrhizic acid decreased 02-”production in U937 cells in a dose dependent manner. However, while caffeic acid, quercetin and curcumin did decrease 02-”production in U937 cells, no dose-dependent effect at the doses examined was apparent (FIG. 3). The ability of these phytochemicals to suppress 02-” production by xanthine/xanthineoxidase was different in some cases to that seen in U937 cells. Chlorogenic acid did not decrease 02-”formation by xanthine/xanthine-oxidase except at the higher concentration tested (500 nM) (FIG. 4). Caffeic acid and quercetin are weak inhibitors of 02-”formation by xanthine/xanthineoxidase. On the other hand, (-)-epicatechin, curcumin and glycyrrhizic acid suppressed 02-’ formation by xanthine/xanthine-oxidase as was also observed in U937 cells. The mechanism of generation of superoxide radicals in the U937 model is completely different to that in the xanthine/xanthine-oxidase model. The difference in behavior of the phytochemicals between the two models may, with fhrther investigation, give some indication of mechanism of how these chemicals inhibit 02-”formation. The ability of phytochemicals to suppress 02-”formation may be due to a direct scavenging action or inhibition of the enzymes involved. The main purpose of this study, however, was to develop, validate and test a cellular model system (U937 cells) for screening the 02-”scavenging ability of phytochemicals. Our findings indicate that U937 cells are a useful model in which to study inhibitors of ‘excess 02-”production. Recent research suggests that many cell types, in addition to phagocytic cells, are capable of producing 02-’when stimulated (38, 43). 02-”and other ROS such hydrogen peroxide, hydroxyl radicals and singlet oxygen have been implicated in a number of degenerative diseases such as cancer and heart disease prevalent in Western societies (44, 45, 46). Consequently, inhibition of ROS generation, including 02-”formation, should provide a measure of protection against such diseases. Research on various food components and naturally occurring compounds capable of reducing the formation and/or scavenging ROS has opened a new approach towards disease prevention. Phytochemicals, such as those examined in this study hold the promise of being usefid disease preventative agents. Further studies using these models will aim to investigate the mechanisms by which phytochemicals modulate 02-”action.

ACKNOWJ.EDCiMRNTS

This project was part-funded by the Department of Agriculture, Food and Forestry, Dublin under the Food Industry Sub-Programmeof EU Structural Funds, 1994-9.

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