Antioxidant Measurement and Applications - American Chemical Society

Chapter 7

Downloaded by KTH ROYAL INST OF TECHNOLOGY on March 1, 2016 | http://pubs.acs.org Publication Date: March 12, 2007 | doi: 10.1021/bk-2007-0956.ch007

Antioxidant Activity of Phytopolyphenols: Assessment in Cell Culture Systems 1

1

Min-Hsiung Pan , Ching-Shu L a i , and Chi-Tang H o

2

1

Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung 811, Taiwan Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, NJ 08901

2

Reactive oxygen species (ROS), are generated during normal physiological processes. ROS are toxic and oxidize of various cell constituents such as D N A , lipids and proteins. The oxidation products so produced may cause damage to cellular machinery, ultimately leading to cell death. ROS have been implicated in a myriad of diseases such as various forms of cancer, atherosclerosis, ischemic reperfusion injury, neurodegenerative diseases, and chronic inflammatory diseases, such as rheumatoid and psoriatic arthritis. Tumor promoters, such as phorbol-12-myristate-13-acetate ( P M A ) enhance the generation of these ROS, through protein kinase C pathway, to activate N A D P H oxidase and xanthine oxidase. Nitric oxide (NO) plays an important role in inflammation and in the multiple stages of carcinogenesis. The suppressive effect of polyphenols on ROS production, monitored by flow cytometry using dichlorodihydrofluorescein diacetate (DCFHDA) and dihydroethidium (DHE), and N O generation are described.

92

© 2007 American Chemical Society In Antioxidant Measurement and Applications; Shahidi, Fereidoon, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

93 Reactive oxygen species (ROS) are broadly defined as oxygen containing chemical species having an unpaired electron or non-radical molecules and these include superoxide anion (0 *~), hydroxyl radical (HO") and hydrogen peroxide ( H 0 ) . In recent years, considerable evidence has emerged implicating ROS as having an important role in the initiation of cellular injury which can lead to cancer development. ROS is not only formed by exogenous sources, but also as a result of normal aerobic metabolism, by neutrophils and eosinophils in their defense against microorganisms and during metabolism of steroids and arachidonic acid. Under normal physiological conditions a balance is maintained between endogenous oxidants and antioxidants, such as superoxide dismutase, catalase and glutathione peroxidase. When an imbalance occurs, created by the excessive generation of oxidants or a decrease of antioxidants, it leads to excessive generation of ROS (1-3). ROS have been implicated in an ever increasing number of diseases and syndromes. It is known that ROS serve as messengers in cellular signaling transduction pathways, and that a moderate increase of certain ROS may promote cellular growth and proliferation and contribute to development of cancer and other diseases (4,5). These include various forms of cancers, atherosclerosis, ischemic reperfusion injury, neurodegenerative diseases and chronic inflammatory diseases, such as rheumatoid and psoriatic arthritis (6-8). Acute and chronic inflammation induced by biological, chemical and physical factors is associated with increased risk of human cancer at various sites. Excessive ROS produced by a variety of inflammatory cells probably participates in the carcinogenic effects of inflammatory reactions. Current evidence indicates that these activated inflammatory cells, induce and activate several oxidant-generating enzymes such as N A D P H oxidase, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) (9-12). These enzymes produce high concentrations of diverse ROS and can cause gene mutation, D N A damage leading to increased mutation and altered functions of enzymes and proteins, thus contributing to the multistage carcinogenesis process thereby being a major factor in tumor promotion (13-15). Tumorigenesis or carcinogenesis is a multistep process that begins with cellular transformation, progresses to hyperproliferation and culminates in the acquisition of invasive potential, angiogenic properties and establishment of metastatic lesions (16). This process can be activated by any one of the various environmental carcinogens, inflammatory agents, tumor promoters and ROS. As described earlier, excessive ROS react with and modify macromolecules resulting either in alterations of D N A structure such as D N A mutations, or in functional modifications of reactive proteins. The biological consequences of ROS are changes in signal transduction, gene expression, and posttranscriptional or post-translational modification that alter cell growth and differentiation and consequently cause carcinogenesis (17,18). In recent years 2


2

2

In Antioxidant Measurement and Applications; Shahidi, Fereidoon, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

94 several studies suggest that ROS also serve an important role as signaling molecules which regulate many genes, including M M P s (19). In biological systems, ROS are constantly generated through a variety of pathways. These ROS can be a primary event in human disease progression or a secondary consequence of tissue injury. Free radicals formed by phagocytes are the first line of immune defense. However, that is accompanied by a dramatic increase in oxygen consumption with the attendant production of 0 ", which is catalyzed by a membrane-bound N A D P H oxidase system in the plasma membrane (20). Even though 0 "~ less reactive than Ό Η , it can react with nitric oxide (NO") to yield an even more reactive species, peroxynitrite that is transformed by dismutates to H 0 and attacks several biological targets (21,22). Lipid peroxidation products hydroperoxy radical ( Ό Ο Η ) , peroxynitrite (ONOO") or hydroxyl radical ( Ό Η ) , have also been implicated in several pathologic conditions including aging, hepatotoxicity, hemolysis, cancer, tumor promotion and inflammation (23,24). Phytopolyphenols, as natural dietary phytochemicals, are widespread in fruits, vegetables, tea, medicinal herbs and other plants. Various phytopolyphenol have long been considered to exert protective effects against many diseases, in particular cardiovascular disease and cancer. The polyphenols might protect the body against cancer and heart disease through inhibition of oxidative damage due to their typical phenolic nature and hydroxyl groups and, therefore, act as potent metal chelators and free radical scavengers (25). Phytopolyphenol actions in vivo or in food may be through inhibiting generation of ROS or increasing the level of endogenous antioxidants. They may also be up-regulated by increased expression of the genes encoding the antioxidant enzymes, such as superoxide dismutase (SOD), catalase or glutathione peroxidase (GPx). Further, phytopolyphenols could also inhibit the source of ROS such as N A D P H oxidase (26,27) and xanthine oxidase (28,29). Oxidative stress and inflammation have been reported as being closely associated with the tumor promotion stage of carcinogenesis. Nitric oxide (NO) is a short lived, highly reactive, free radical which is produced from L-arginine by nitric-oxide synthase. The overproduction of N O is thought to contribute significantly to the pathogenesis of inflammatory demyelinating diseases, such as multiple sclerosis, and neurodegeneration in certain diseases such as Alzheimer's and Parkinson's. Furthermore, N O and its oxidized forms have also been shown to be carcinogenic (30,31).


2

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2

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Materials and Methods Cell Lines The human gastric carcinoma cell line A G S , leukemia cell line HL-60 and mouse macrophage RAW264.7 (American Type Culture Collection [ATCC])



95 were respectively cultured in Dulbecco's modified Eagle's F12, Roswell Park Memorial Institute 1640 and Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. The human A G S gastric carcinoma cell lines (CCRC 60102) were obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cell lines were grown at 37 °C in 5% carbon dioxide atmosphere in Dulbecco's modified Eagle's medium for RAW264.7 cells; D M E M / F 1 2 for A G S cells; and RPMI for HL-60 cells, supplemented with 10% heat-inactivated fetal bovine serum (Gibco B R L , Grand Island, N Y , 100 units/mL of penicillin, 100 μg/mL of streptomycin), and 2 m M 1-glutamine (Gibco BRL).

Measurement of Cellular G S H Content by Flow Cytometry The level of intracellular G S H per cell was determined by flow cytometry after staining with chloromethylfluorescein-diacetate (CMF-DA) (32). C M F - D A , containing a mild thiol reactive chloromethyl reactive group, is colorless and nonfluorescent. This probe is primarily conjugated to the abundant tripeptide glutathione by glutathione S-transferase. Once inside the cell, cytosolic esterases cleave off their acetates and then the chloromethyl group reacts with intracellular thiols, transforming the probe into a cell-impermeant fluorescent dye-thioether adduct. In this experiment, A G S cells were treated with 60 μΜ acacetin for 0.5, 1 and 2 h, after which C M F D A (final concentration was 25 μΜ) was added into the medium. The cells and fluorescent dyes were incubated for 30 min at 37 °C. After 30 min, cells were washed with PBS, and the intracellular G S H levels were determined by flow cytometry.

0 "~ Production Determination 2

To detect XO/xanthine-induced intracellular ROS accumulation, HL-60 cells were treated with various flavonoids (luteolin, apigenin, chrysin, diosmetin, acacetin, nobiletin and tangeretin) for 30 min prior to xanthine oxidase (9 U/L) / xanthine (50 μΜ) treatment for 30 min. The cells were washed once with PBS, and D H E (20 μΜ) was added into the medium for a further 30 min. The superoxide anion ( 0 · - ) production was monitored by flow cytometry. 2

H 0 -Induced Oxidative Cell Damage 2

2

5

HL-60 cells (2 xlO ) were pretreated with test compounds for 30 min prior to the addition of 50 μΜ H 0 into the medium for a further 8 h. The cells were then harvested, washed with PBS, resuspended in 200 μΐ, of PBS, and fixed in 2

2


96


800 μι. of iced 100% ethanol at -20 °C. After being left to stand overnight, the cell pellets were collected by centrifugation, resuspended in 1 mL of hypotonic buffer (0.5% Triton X-100 in PBS and 0.5 μg/mL RNase) and incubated at 37 °C for 30 min. Next, 1 mL of propidium iodide solution (50 μg/mL) was added and the mixture was allowed to stand on ice for 30 min. Fluorescence emitted from the propidium iodide-DNA complex was quantitated after excitation of the fluorescent dye by F A C Scan cytometry (Becton Dickinson, San Jose, CA).

DNA Extraction and Electrophoresis Analysis HL-60 cells were treated, with or without flavonoids, in the presence of xanthine oxidase (9 U/L)/xanthine (50 μΜ) for 4 h. The cells were then harvested, washed with phosphate-buffered saline (PBS) and lysed with digestion buffer containing 0.5% sarcosyl, 0.5 mg/mL proteinase K , 50 m M tris(hydroxymethyl) aminomethane (pH 8.0) and 10 m M E D T A at 56 °C overnight after which they were treated with RNase A (0.5 μg/mL) for 3 h at 56 °C. The D N A was extracted by phenol/chloroform/isoamyl (25:24:1, v/v/v) before loading and was analyzed using 2% agarose gel electrophoresis. The agarose gels were run at 50 V for 120 min in Tris-borate/EDTA electrophoresis buffer (TBE). Approximately 20 μg of D N A was loaded into each well, visualized under U V light and photographed (33).

Measurement of Intracellular R O S Accumulation T P A is an inflammatory agent and potent tumor promoter which has been reporter to act through the generation of ROS. To investigate whether polyphenols could reduce TPA-induced ROS production, HL-60 cells were pretreated with test compounds for 30 min prior to T P A (100 ng/mL) treatment for a further 30 min. The intracellular ROS levels in HL-60 cells were detected by 2',7'-Dichlorodihydrofluorescein diacetate (DCF-DA). D C F - D A is hydrolyzed by cellular esterases to firm DCF, thus, the fluorescence intensity is proportional to the amount of peroxide produced by the cells.

Nitrite Assay Mouse macrophage RAW264.7 cells were treated with test compounds and LPS (100 ng/mL) for 24 h. The nitrite concentration in the medium was measured as an indicator of N O production according to the Griess reaction. One hundred microliters from each supernatant was mixed with the same volume of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1%


97 naphthylethylenediamine dihydrochloride in water) and absorbance of the mixture was'read at 550 nm using an ELISA plate reader (Dynatech MR-7000; Dynatech Laboratories).

Results Effect of Luteolin and Apigenin on H 0 Production of TPA-stimulated HL-60 Cells Downloaded by KTH ROYAL INST OF TECHNOLOGY on March 1, 2016 | http://pubs.acs.org Publication Date: March 12, 2007 | doi: 10.1021/bk-2007-0956.ch007

2

2

The effects of luteolin and apigenin on H 0 production by TPA-stimulation HL-60 cells were analyzed by flow cytometry. The results showed that luteolin was a potent inhibitor of TPA-induced H 0 production in HL-60 cells at 10 μΜ (Figure 1). The inhibitory potency was luteolin > apigenin at 10 μΜ (Figure 2). The effects of luteolin on H 0 production in TPA-stimulated HL-60 cells are possibly due to involvement in the signal transduction of the P K C activation pathway that is stimulated by TPA. 2

2

2

2

2

2

Effects of Flavonoids on H 0 Levels in HL-60 Cells 2

2

The H 0 levels was further compared in only flavonoid (10 μΜ) treated HL-60 cells using the fluorescent probe D C F H - D A and monitoring by flow cytometry. A decrease of intracellular peroxide levels by 10 μΜ tangeretin was detected for 0.5 h. The inhibitory potency was estimated in the following order: tangeretin > nobiletin > apipenin > luteolin > chrysin > diosmetin at 10 μΜ (Figure 3). 2

2

Effect of Flavonoids on H O i n d u c e d Apoptosis in HL-60 Cells 2

r

Hydrogen peroxide which has a permeable cell membrane and is a precursor of various free radicals, was chosen as an oxidant to induce apoptosis in our study. To further assess the antioxidant activity of flavonoids, we investigated whether exogenously administered H 0 could induce apoptosis in HL-60 cells. As shown in Figure 4, luteolin (10 μΜ) proved to be an efficient protective agent for H 0 -induced apoptosis in HL-60 cells. 2

2

2

2

Inhibition of ROS Generation and Protection of HL-60 Cells from ROS Induced Apoptosis by Flavonoids Xanthine oxidase (XO) is a complex enzyme. It causes gout and is responsible for oxidative damage to living tissues. X O is the major source of


98


δ

R.1-H

ι*

Figure 1. Effects of Luteolin on TPA-induced hydrogen peroxide (H2O2) generation in HL-60 cells. Cells were treated TPA only or treated with 10 μΜ luteolin prior to TPA (100 ng/mL) treatment.Tthe cells were then incubated with DCFH-DA (20 μΜ) for 30 min and analyzed by flow cytometry and presented as log fluorescence intensity. A, control, B, TPA 100 ng/mL + luteolin 10 μΜ, C, TPA WOng/L.

ROS candidacy, as strengthened by recent analyses of promoter regions of human, mouse, and rat enzymes, which suggest the presence of potential regulatory sites for cytokines known to stimulate generation of ROS. Flavonoids are known to inhibit xanthine oxidase activity and scavenge ROS. The flavonoid prevention of ROS generation induced by external xanthine/xanthine oxidase (X/XO) reaction was analyzed. The HL-60 cells were treated with xanthine and xanthine oxidase, and ROS generation and cell apoptosis were assayed by flow cytometry and D N A electrophoresis. The inhibitory ability of ROS generation by flavonoids is as follows: chrysin > nobiletin > diosmetin > luteolin > apigenin > tangeretin > acacetin at 10 μΜ (Figure 5). As shown in Figure 6, the D N A ladder was prevented when cells were treated with flavonoids. These results suggest that flavonoids significantly protected cells from ROS induced apoptosis in the X / X O reaction. These flavonoids were able to prevent cell damage induced X / X O reaction and could be potent inhibitor of xanthine oxidase.


99

DCFH-DA


10°

10

1

IO

to

2

io

3

ttì°

4

10

1

10°

10

1

10

2

C

to

2

to

3

to

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10

3

10

4

ΨΙΡΗ

FU*H

1Ô

3

10

·

4

10°

to

Luteolin

1

10 FL1-H 2

Apigenin

(10 μΜ)

TPA (100 ng/ml)

Figure 2. The effect of Luteolin and Apigenin on TPA-induced hydrogen peroxide (H 0 ) production in HL-60 cells. Cells were treated TPA only or treated with 10 μΜ luteolin and apigenin prior to TPA (100 ng/mL) treatment. The cells were then incubated with DCFH-DA (20 μΜ) for 30 min and analyzed by flow cytometry and presented as log fluorescence intensity. 2

2



100

Figure 3. The effect of flavonoids on hydrogen peroxide (H 0 ) levels in HL-60 cells. Cells were treated with 10 μΜΑανοηίά5 (1, apigenin; 2, luteolin; 3, chrysin; 4, diosmetin; 5, acacetin; 6, nobiletin; 7, tangeretin) 30 min and were then incubated with DCFH-DA (20 μΜ) for a further 30 min. Data are analyzed by flow cytometry and presented as log fluorescence intensity. 2

H 0 2

2

2

(50 μΜ)

Figure 4. Effects of various flavonoids on H 0 -induced apoptosis in HL-60 cells. HL-60 cells were pretreated 10 μΜflavonoids (1, apigenin; 2, luteolin; 3, chrysin; 4, diosmetin; 5, acacetin; 6, nobiletin; 7, tangeretin) for 30 min prior to treated with 50 μΜΗ 0 ^ 8 h. The apoptotic ratio (%) was determined by flow cytometry. 2

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140

Xanthine / Xanthine Oxidase

Figure 5. Effect offlavonoid on Relative percent of reactive oxygen species (ROS) in xanthine /xanthine oxidase-induced ROS production in HL-60 cells. Cells were treated with 10 μΜΑανοηίά5 (1, apigenin; 2, luteolin; 3, chrysin; 4, diosmetin; 5, acacetin; 6, nobiletin; 7, tangeretin) for 30 min, followed by incubated with xanthine/xanthine oxidase for a further 30 min. The cells were then incubated with DHE (20 μΜ) for 30 min and analyzed by flow cytometry and presented as log fluorescence intensity.

Inhibition of NO Generation by Polyphenols in LPS-activated Macrophages Nitric oxide plays an important role in inflammation and in the multiple stages of carcinogenesis. O f the polyphenols tested, theaflavin-3,3'-digallate (20 μΜ) inhibited LPS-stimulated N O generation the most strongly. As shown in Table I, the inhibitory potency was estimated to be in the following order: theaflavin-3,3'-digallate > acacetin > wogonin > penta-O-galloyl-P-D-glucose at 20 μΜ.

Discussion Reactive oxygen species (ROS) are a family of active molecules including superoxide anion (0 "~), peroxyl (ROO'), hydroxyl radical (HO"), and nitric oxide (NO), that are generated in cells by several pathways and involved in the 2


102

Xanthine / Xanthine Oxidase


M - 1

2

3

4

5

6

7

8

Figure 6. Flavonoids protected HL-60 cells from ROS damage in the xanthine oxidase/xanthine reaction. HL-60 cells treated with or without Flavonoids in the presence ofxanthine oxidase/xanthine for 4h, and DNA fragmentation was shown. (1, apigenin; 2, luteolin; 3, chrysin; 4, diosmetin; 5, acacetin; 6, nobiletin; 7, tangeretin)

modulation of biological cell functions. However, a large amount or sustained levels of ROS can result in the oxidation of biomolecules including lipid, protein, and D N A , resulting in cell damage leading to growth arrest, senescence, or death (34). Several studies have shown that differentiated HL-60 cells possess the phagocytic properties and the capability of generating ROS upon stimulation (35). Human carcinogenesis is known to progress through multiple stages of initiation, promotion, and progression. It is well known that generation of ROS is associated with the initiation and promotion of carcinogenesis. Chemoprevention has had a potential impact on cancer incidence rates through the modulation of initiation and promotion stages (36). Epidemiological studies have increasingly demonstrated that the content of phytochemicals such as curcumin, tea polyphenols, and flavonoids occur ubiquitously in plant foods. They reduce the risk of cancer through their role in the metabolism of carcinogens, in hormonal binding, in regulation of gene expression, in antioxidant enzymatic activities, and in scavenging of free radicals (37). The purpose of this study was to evaluate antioxidant activities of phytophenols in different cell culture assay for the prevention of carcinogenesis.


103 Table 1. Effects of Phytopolyphenols on LPS-induced Nitrite Production in RAW 264.7 Macrophage

control LPS (100ng/ml)

-

LPS

Wogonin

10 20

Nitrite (μΜ) 4.6±0.6 43.3±0.9 23.1±0.4 17.1±0.6

LPS

Acacetin

10 20

14.6±0.3 8.2±0.5

74 91

LPS

5GG

10 20

24.7±0.9 18.3±0.6

48 65

LPS

TF-3

10 20

13.2±0.4 6.8±0.2

78 94

Test Compounds (μΜ)


Sample

Inhibition (%) 52 68

The cells were treated with 100 ng/ml of LPS only or with different concentrations (10 and 10 μΜ) of various phytopolyphenols for 24 h. At the end of incubation time, 100 μΐ of the culture medium was collected for nitrite assay

Because oxidative D N A damage is considered to be relevant in carcinogenic processes, we evaluated the possible anticarcinogenic effects of flavonoids by determining their effect on TPA-inducing ROS generation, H 0 scavenging, H 0 -induced apoptosis, xanthine oxidase activity, and LPS-inducing N O generation. Assays of various antioxidant models produced different effects. Luteolin inhibited the H 0 production of TPA-stimulated HL-60 cells (Figure 1 and 2). Tangeretin more potent removed the level of H 0 in HL-60 cells (Figure 3). T P A is known to induce H 0 production by phagocytic cells and epidermal cells through increasing X O activity and by diminishing antioxidant enzyme activities (38). Recent studies show that, in double T P A treated mouse skin, ROS from leukocytes, including superoxide, plays an important role leading to chronic inflammation and hyperplasia. Superoxide generation inhibitors are effective in inhibiting this tumor promotion response. These findings might suggest that phytopolyphenols act at an earlier stage than has previously been suspected, by suppressing ROS production through inhibiting X O , rather than only scavenging the already formed ROS. This could partly explain some of the beneficial properties attributed to phytopolyphenols, such as antimutagenic and anticarcinogenic effects which are all mediated by ROS (39). 2

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Acknowledgements This study was supported by the National Science Council Grant N S C 942321-B-022-001.


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