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Role of Digestion, Iron and Protein on the Antioxidant
Effect of Green and Black Tea in CacoZ Cells

presented by
Shama Vasanthi Joseph

has been accepted towards fulﬁllment
of the requirements for

M.S. Jegeemmmition

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Major professor

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6/01 cJCIRCJDaIeDuopBS-p. 15

ROLE OF DIGESTION, IRON AND PROTEIN ON THE ANTIOXIDANT EFFECT
OF GREEN AND BLACK TEA IN CAC02 CELLS

By

Shama Vasanthi Joseph

A THESIS

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Food Science and Human Nutrition

2002

ABSTRACT

ROLE OF DIGESTION AND POLYPHENOL-NUTRIENT INTERACTION ON
ANTIOXIDANT EFFECT OF GREEN AND BLACK TEA IN CAC02 CELLS

By
Shama Vasanthi Joseph

Tea made from the leaves of Camellia sinensis has been vigorously investigated
for its antioxidant and potential anti-cancer effects. Most studies have shown beneﬁcial
effects of its puriﬁed extracts or individual constituents. Evidence on the inﬂuence of
digestion and other dietary components during digestion is sparse. The aim of this study
was to elucidate the effect of simulated digestion on the antioxidant effect of green and
black tea and the inﬂuence of iron and protein during this process. CacoZ cells were pre-
treated for 1 h with either a black or green tea decoction (4 g in 50 ml boiling water for 5
min). digested tea decoction. or tea digested with cereal or with milk and then treated
with 500 uM hydrogen peroxide (H202). DNA damage was estimated as single strand
breaks (Comet assay) and as 8-OHdG formation. Pre-treatrnent with green tea decoction
signiﬁcantlyreduced the percentage of comets from 52 to 36% in the comet assay.
However, H203 treatment did not cause signiﬁcant level of DNA damage in the
8-OHdG assay compared to untreated cells (control) thus making it difﬁcult to make any
conclusions related to a protective effect of pre-treatment with tea or tea digest. Neither
green nor black tea caused a signiﬁcant change in 8—OHdG content of cells compared to
H202 treatment alone. The digestive process had no effect nor did the addition of cereal
or milk, except for an apparent pro-oxidant effect with the addition of milk to green tea.
Milk and cereal interacted differently with green and black teas. as shown by signiﬁcantly
higher concentrations of 8-OHdG when each of these foods was digested with green tea

compared to black tea.

ACKNOWLEDGEMENTS

Achieving anything in life would not mean much if I did not acknowledge my
Lord and Savior Jesus Christ due to whose grace this project was brought to its successﬁil
completion.

My heartfelt gratitude goes out to my major advisor Dr. Wanda Chenoweth for
her unfailing support, guidance and enthusiasm throughout this program. I was
especially touched by her sincere interest in my welfare. I thank her for her selﬂess help
and for inspiring me to persevere in the face of all odds.

Many thanks are due to my committee members Drs. Bennink and Hord who
provided expert technical advice as well as their constant support and encouragement
during my studies.

I offer my special thanks to Dr. Gangur for his generosity in lending me
invaluable technical guidance for this project. I am also thankful to Drs. Weatherspoon
and Strasburg for permitting me to use their lab facilities.

To all my friends, I owe my thanks for their amazing moral support and genuine
friendship. To Deepa, who has been my closest friend and an inspiration to succeed, I
will always be grateful. To Winnie, who patiently helped me with lab techniques and
constantly encouraged me to do my best, I offer my gratitude. My sincere thanks go out
to Lisa, Ross and Sandhya for being true friends and for readily offering their help in
times of need. I also thank Auntie and Uncle Rao for their prayers and generous
hospitality.

This acknowledgement would not be complete without offering my thanks to my

wonderful parents, Richard and Vimala Joseph, to whom I owe everything. I also thank

iii

my sisters Suman and Vanya and my brother-in-law Madhu for the unbelievable love,

encouragement and moral support that kept me going throughout this program.

iv

TABLE OF CONTENTS

LIST OF TABLES ........................................................................... vii
LIST OF FIGURES ......................................................................... viii
INTRODUCTION ........................................................................... 1
REVIEW OF LITERATURE ............................................................. 3
Dietary polyphenols
Chemistry and bioavailability ............................................. 3
Polyphenol-nutrient interactions .......................................... 5

Polyphenols in chronic disease prevention

Cancer
Oxidative damage in etiology of cancer ................................. 6
Mechanism of oxidation ................................................... 7
Oxidative DNA damage in cancer ........................................ 8
Antioxidant character of polyphenols .................................... 8
Prooxidant character of polyphenols ..................................... 10
Cardiovascular disease ............................................................. ll

Importance of polyphenolic compounds in tea

Use and composition of tea ................................................ 12

Anti-cancer effects of tea ................................................... 13
Epidemiological and clinical studies ..................................... 16

In vitro and animal studies ................................................. 17
Anti-angiogenic effects of catechins ..................................... 18

Food matrix eﬂects ......................................................... 19
JUSTIFICATION AND OBJECTIVES ................................................ 21
HYPOTHESIS ............................................................................... 21
MATERIALS AND METHODS ......................................................... 22
Research design ...................................................................... 21
Materials .............................................................................. 21
Chemicals ............................................................................. 24

Cell culture ............................................................................ 25

Cell viability .......................................................................... 26
Hydrogen peroxide oxidation: preliminary experiments ....................... 26
Simulated digestion .................................................................. 26
Treatment of cells .................................................................... 27

Estimation of DNA damage by Alkaline single cell gel

electrophoresis (Comet assay) ............................................. 30
Estimation of DNA damage by formation of

8-Hydroxy-2’-deoxyguanosine (8-0HdG)

DNA isolation ............................................................... 3 1
DNA digestion ............................................................... 31
8-0HdG assay ............................................................... 33
Statistical analysis .................................................................... 35
RESULTS
Effect of H202 on viability of Cac02 cells and formation of
DNA single strand breaks .................................................. 36
Effect of H202 on DNA single strand breaks in Cac02 cells .................. 37
Effect of green tea pre-treatment on DNA single strand
breaks in Cac02 cells ........................................................ 37
Production of 8-Hydroxy-2'-deoxyguanosine (8-0HdG)
Eﬂect 0f500/1M H202 ...................................................... 40
Effect of pre-treatment with green tea .................................... 4O
Eﬂect of pre-treatment with black tea .................................... 43
DISCUSSION ................................................................................. 46
SUMMARY AND CONCLUSIONS ..................................................... 55
SUGGESTIONS FOR FUTURE RESEARCH ........................................ 58
BIBLIOGRAPHY ........................................................................... 60

vi

LIST OF TABLES

. Polyphenolic components of green and black teas as % dry
weight of solids ........................................................................... 15

. Nutrient content of 2 % milk and Product l9® breakfast cereal .................. 23

. Concentration of 8-hydroxy—2'-deoxyguanosine (8-0HdG) in

Cac02 cells pre-treated with green tea decoction or tea

decoction digested by itself or with milk or cereal followed

by treatment with SOOuM H202 ....................................................... 41

. Concentration of 8-hydroxy-2'-deoxyguanosine (8-0HdG)

in Cac02 cells pre-treated with black tea decoction or tea

decoction digested by itself or with milk or cereal followed

by treatment with SOOuM H202 ....................................................... 42

vii

LIST OF FIGURES

. Structure of tea polyphenols ......................................................... l4
. Flow diagram of experimental design ............................................. 29
. Gel electrophoresis of DNA and digested DNA of Cac02 cells ............... 32

. Standard curve of 8-hydroxy-2'-deoxyguanosine (8-0HdG) by
using the Highly Sensitive 8-0HdG kit ........................................... 34

. Effect of hydrogen peroxide and green tea treatment on
percentage of comets formed in Cac02 cells ..................................... 38

. Effect of hydrogen peroxide and green tea on tail length of
comets formed in Cac02 cells in a typical experiment .......................... 39

. 8-0HdG content in Cac02 cells pre-treated with green or black

tea decoction (4 g tea leaves in 50 ml boiling water) or tea

digests, followed by hydrogen peroxide treatment (500 uM) for

20 min. ............................................................................... 44

viii

INTRODUCTION

Substantial epidemiological data indicates that an inverse relationship exists
between a fruit and vegetable rich-diet and risk for certain chronic diseases. Free radicals
have been implicated in the etiology of diseases such as cancer and cardiovascular
disease. DNA damage due to oxidation is particularly signiﬁcant in the causation of
cancer. Oxidative damage as an etiological factor in such diseases occurs when there is
an imbalance between oxidants and anti-oxidants. A wide body of literature suggests that
the efﬁcacy of dietary antioxidants depend largely on the extent to which they are
absorbed and metabolized in order to exert this effect. However only recently has it been
highlighted that antioxidant activity might be required at the site of absorption itself, i.e.,
the intestine. This portion of the digestive system is exposed to various toxic compounds
including carcinogens indicating that it requires to be protected against oxidative attack,
which could potentially lead to cancer. (Halliwell et a1, 2001).

Most plant tissues contain a wide variety of secondary metabolites such as
alkaloids, terpenes, rare amino acids, steroids and polyphenols, of which polyphenols
have received recent and widespread attention owing to their health beneﬁts.
Polyphenols are an extremely heterogeneous group of natural products comprising
various sub-groups including the ﬂavonoids. It is now well established that the non-
nutrient polyphenolic components of various plants, including tea, which possess free
radical scavenging and metal sequestering properties and thereby antioxidant capacity are
responsible in part for the phenomenon observed in epidemiological studies.

Intensive research in the past decade has suggested tea derived from the plant
Camellia sinensis to play a protective role against cancer of different sites. The evidence

has been derived from and conﬁrmed mostly by in vitro and animal studies but

epidemiological evidence has been equivocal at best. The anticancer activity of tea has
been attributed to its polyphenolic constituents namely catechins. Of these compounds
epigallocatechin gallate (EGCG), the largest fraction, has been demonstrated to be mainly
responsible for these effects (Yang et a1, 2002).

Once considered anti-nutrients, plant polyphenols are known to interact and bind
with other nutrients particularly protein and transition metal ions. These compounds have
a high afﬁnity for proline-n'ch proteins such as casein. Polyphenols not only reduce
digestibility of proteins but also bind enzymes rendering them inactive. Tea polyphenols
have long been known to form insoluble complexes with iron in the gastrointestinal tract.
The absorption of non-heme iron but not heme iron is thereby inhibited. This effect can,
however, be overcome by the presence of ascorbic acid (Dufresne et al, 2001).

Literature on the beneﬁcial effects and mechanistic aspects of dietary polyphenols
is proliﬁc. However, little is known about'how the antioxidant effects are inﬂuenced
when polyphenolic compounds are consumed in conjunction with other dietary
components. 0f the different polyphenolic compounds studied, those from green and
black teas have been the focus of much attention, although studies have primarily
involved employing concentrations many fold greater than what would be possible
physiologically. The present study was designed to further elucidate the antioxidant effect
of green and black tea as part of a ‘meal’, taking into consideration polyphenol-metal and
polyphenol-protein interactions against a backdrop of physiologically plausible

concentrations of these compounds.

REVIEW OF LITERATURE
Dietary polyphenols

Chemistry and bioavailability. Polyphenols are a group of non-nutrient
secondary metabolites present ahnost ubiquitously in plant organs. They are synthesized
via the shikimate pathway. Initially considered anti-nutrients due to their deleterious
ability to chelate metals such as iron in addition to carbohydrates and proteins (Hulse et
al., 1980), they now receive widespread interest for their antioxidant properties. Natural
polyphenols exist in various forms ranging ﬁom simple monomers like phenolic acids to
complex polymers like tannins. They occur mainly in conjugated form with sugar
residues linked to hydroxyl groups. The sugars are generally present as
monosaccharides, disaccharides and oligosaccharides. Polyphenols can be classiﬁed into
at least 10 different groups based on their chemical structure (Harborne, 1989).
Flavonoids are the largest class of polyphenols comprising about 5000 compounds. They
are further classiﬁed into ﬂavones, ﬂavanols, isoﬂavones, ﬂavonols, ﬂavanones and
anthocyanins. Tea polyphenols are ﬂavanols and are comprised mainly of catechin,
epicatechin and epigallocatechin.

Intake of 'dietary polyphenols has been estimated to be close to 1 g/day (Scalbert
and Williams, 2000). However, limited data are available on the absorption and
biological action of these compounds. Some studies have demonstrated that polyphenolic
compounds and their metabolites are detectable in plasma after ingestion (van het Hof et
al., 1998, Erlund et al., 2000). Other studies have assessed their digestive fate by urinary
excretion of these compounds (Young et al., 1999). For the most part, data have been

obtained from research that used plant extracts or pure standards (Okabe et al., 1999).

Therefore, differences are to be expected in uptake and subsequent metabolism when
polyphenols are consumed as part of a complex food matrix.

Little is known about the effect of the gastric milieu and the digestive process on
the absorption of dietary polyphenols. Results from an in vitro study by Spencer et a1.
(2000), using simulated pepsin-free gastric juice showed that under gastric conditions
procyanidin oligomers of catechins are hydrolyzed to a mixture of epicatechin dimers and
monomers, thus increasing their potential for absorption by the small intestine. Some
early animal studies showed that many common dietary ﬂavonoids and aglycones are
only partially absorbed due to microbial destruction of the heterocyclic rings of the
compounds before any absorption occurs in the small gut (Duthie and Dobson, 1999). In
a study in which quercetin and catechin were shown to be absorbed by the rat intestine,
the plasma and liver levels of the two compounds were increased with consumption of
diets supplemented with these compounds. The compounds were present as conjugated
derivatives in the plasma. The plasma concentration of catechin metabolites was seen to
drop dramatically in the post-absorptive period. This decrease was not observed with
quercetin derivatives (Manach et al., 1999).

Epicatechin and theobromine uptake from black chocolate was assessed in an
intervention study with 8 healthy males. Plasma concentration of both compounds
increased signiﬁcantly after consumption of chocolate, reaching a maximum between
2-3 h. Their study also revealed that although epicatechin is absorbed from chocolate, it
is rapidly eliminated from plasma (Richelle etal., 1999). Cao and group (1998) used the
Oxygen Radical Absorbing Assay (ORAC) to evaluate changes in plasma antioxidant
capacity resulting from two diets in 36 healthy human subjects. The ﬁrst diet included 10

servings of fruits and vegetables per day. Plasma antioxidant capacity as oxygen radical

antioxidant capacity (ORAC) and a-toc0pherol levels were measured in subjects. The
second diet was comprised of the ﬁrst diet with the addition of two servings of broccoli.
Results indicated that there was a signiﬁcant elevation of plasma antioxidant capacity
compared with fasting levels for both the diets, which could not be accounted for by
a-tocopherol.

In a study to evaluate bioavailabilty of gallic acid from black tea in humans,
Sharzad et a1. (2001) reported that gallic acid from black tea is rapidly absorbed and
eliminated with a mean half life of 1.06 i- 0.06 h and a mean maximum plasma
concentration of 2.09 i 0.22 umoles /L. 0f the gallic acid dose (200 ml brewed tea with
50mg free gallic acid from the tea), 39.6 i 5.1% was excreted in urine in the form of
gallic acid and its metabolite 4-0-methylgallic acid. In yet another study of polyphenol
bioavailability, 9 human subj ects ingested a single moderate serving (120ml) of de-
alcoholized red wine reconstituted with either water (DRW) or alcohol (ARW). No
difference was obServed in total plasma (+)-catechin concentration with either the DRW
or ARW. Alcohol signiﬁcantly increased the elimination rate of (+)-catechin from the
plasma suggesting either excretion of the compound or its conversion to methylated
derivatives (Bell et al., 2000). Boyle and colleagues (2000) studied the effect of rutin
supplementation in 18 healthy female volunteers. Six weeks of rutin supplementation
signiﬁcantly elevated plasma levels of quercetin, kaempferol and isorharnnetin.
However, no difference was seen in the plasma antioxidant status of these subjects
measured by Ferric Reducing Ability of Plasma ( F RAP) assay.

PolyphenoI-nutrient interactions. Polyphenols have long been known to interact

with other dietary compounds. They exhibit a strong afﬁnity for proline-rich proteins

such as casein, gelatin and salivary proteins. Large polyphenols like tannins, which are
poorly soluble in water, show the most effect in this regard. Complexes with proteins are
formed through multiple hydrogen bonds between hydroxyl groups of tannins and the
carboxyl function of the peptide linkages of protein (Loomis and Bataille, 1966). One
molecule of tannin binds two or more protein chains resulting in precipitation. This
property of polyphenols might reduce the digestibility of alimentary proteins and increase
fecal nitrogen output. Tannins also complex digestive enzymes trypsin, amylase and
lipase thereby reducing digestibility of starch, protein and lipids.

Polyphenols interact strongly with transition metal ions. They form insoluble
complexes with iron in the gastrointestinal tract. Inhibition of dietary iron absorption has
been shown to occur due to this phenomenon. In studies with tea polyphenols, black tea
inhibited iron absorption more strongly than green tea. It is to be noted, however, that
this property has bearing only on non-heme iron and not that derived from animal
sources. Furthermore, this deleterious effect on absorption may be overcome by the

simultaneous presence of ascorbic acid in the diet (Duﬁesne et al., 2001).

Polyphenols in chronic disease prevention
Cancer
Oxidative damage in etiology of cancer. Because of their ability to act as
antioxidants, much attention has focused on a possible role of polyphenolic compounds
in prevention of cancer. Studies on the causes of various cancers including that of the
colon have identiﬁed oxidative cellular damage as one of the prime factors in
tumorigenesis. Dietary antioxidants, in addition to endogenous antioxidant enzyme

systems like glutathione peroxidase and catalase, confer protection against the onslaught ~

of oxidation mediated by reactive oxygen species (ROS) and other free radical species
(by-products of the normal metabolic processes of the body).

Cancer development is a multi-stage process involving reactive oxygen species
as well as other free radicals in its initiation as well as progression (Bravo, 1998). It has
been suggested that the gut is the primary site of ROS assault as well as the ﬁrst organ to
be associated with ingested nutrients including antioxidant phytochemicals. It is
therefore logical to conclude that a major proportion of anti-cancer activity of dietary
antioxidants is being exerted at the site of contact in the intestine (Halliwell et al., 2001).

Mechanisms of oxidation. Oxidation products are generated as a natural part of
the daily metabolic processes of the cell. They are highly reactive molecules and include
hydroxyl radical, superoxide anion, peroxyl radical, nitric oxide, nitrogen dioxide,
peroxynitrite and singlet oxygen. A highly regulated balance exists between oxidants and
antioxidants. Oxidative damage results from a shift in this balance due to overproduction
of reactive oxygen species leading to disruption of cellular functions. Particularly
relevant is the fact that oxidation of nucleic acid bases in DNA results in mutations and
increase the risk of cancer occurrence.

An important route to active oxygen species is the Fenton reaction (equations 1
and 2).

O2" +Fe (III)-)O2+ Fe (II) (1)

Fe (II) + H20 2 -) Fe (III) + 110' + H0 ' (2)
Free radicals are detoxiﬁed by a multi stage enzyme system in which the molecules are
activated by phase I enzymes (cytochrome P450 and NADPH) converting them into

electrophilic, water-soluble compounds. Thereafter they are conjugated to detoxifying

molecules like glutathione and amino acids for inactivation and excretion (Dufresne et
aL,2001)

Oxidative DNA damage in Cancer. Damage to DNA resulting from oxidation
can possibly lead to the development of cancer. Several biomarkers have been identiﬁed
and methods have been developed and used to measure DNA damage. Of these 8-
hydroxy-Z’ deoxyguanosine (8-0HdG) is a DNA base modiﬁed product generated by
reactive oxygen species (ROS). This oxidized base has been widely utilized and is an
established and well-accepted biomarker of damage produced by oxygen radicals. The
C-8 position of guanine is oxidized to give a mutagenic lesion leading to mainly G-T
transversion mutations (Halliwell, 2000). Inagake et a1. (1995) showed that in rats treated
with 1,2-dimethyl hydrazine the production of 8-OHdG was signiﬁcantly inhibited in the
colon thereby protecting the colonic mucosa from oxidative damage. Hasegawa and
colleagues (1995) fed 2 % green tea as drinking water for two weeks to male rats treated
intra-peritoneally with 2-nitropropane. As a result liver nuclear 8-OHdG concentration
was signiﬁcantly reduced.

Antioxidant character of polyphenols. Evidence on cellular mechanisms by
which dietary polyphenols may offer protection against carcinogenesis is controversial
and not entirely clear. However, their antioxidant properties have been studied most
intensively. In a study by Aheme and O’Brien (2000), quercetin and rutin offered
protection against tert-butylhydroperoxide induced DNA strand breaks in Cac02 cells.
Only quercetin prevented this damage when menadione was employed as the pro-oxidant.
This effect was due to its action as both a metal chelator as well as radical scavenger.
Russo and colleagues (2000) showed that the bioﬂavonoids rutin, catechin and naringenin

had a cell protective effect by scavenging free radicals demonstrated by their ability to

bleach 1,1-diphenyl-2-picrylhydrazyl radical. These compounds also inhibited lipid
peroxidation in rat liver microsomes and xanthine oxidase activity. In addition they also
protected pBR322 plasmid DNA from oxidative cleavage.

Another study conducted to elucidate protective mechanism of polyphenols, Kaul
and Khanduja (1999) showed that ellagic acid, tannic acid, caffeic acid and ferulic acid
protected murine peritoneal macrophages from superoxide anion radicals (SOR)
generated by the tumor promoter benzoyl peroxide (BOP). The protective effect was due
to inhibition of SOR formation and ferulic acid was shown to have the greatest effect.
These compounds also inhibited diacylglycerol accumulation and concurrent choline
equivalent release caused by the BOP. Duthie and Dobson (1999) investigated the
protective effect of select ﬂavonoids against hydrogen peroxide (H202) induced single
strand breaks in DNA of human colon cancer cells. Their ﬁndings pointed to a beneﬁcial
effect of myricetin and quercetin, the most abundant ﬂavonoid in the human diet,
whereas rutin and kaempferol were not shown to be effective antioxidants. Quercetin did
not alter the level of oxidized bases even though it reduced DNA strand breakage. Their
study also suggested that P450-mediated metabolism may be involved in the efﬁcacy of
these compounds. . _

Casalini et al. (1999) fed rats treated with 2-nitropropane a mixture of wine
complex polyphenols and tannins (WCPT). The dose, 57 mg/kg for 14 d, was ten fold
higher than what a moderate wine drinker would consume. There was a signiﬁcant
lowering of the ratio of 8-hydroxy-2’-deoxyguanosine to 2-deoxyguanosine, a marker of
DNA damage, compared to controls. However, pre-treatment with WCPT for 10 days
did not protect colonic'mucosa from oxidative DNA damage induced by

1,2-methyldihydrazine. The antioxidant potencies of several widespread ﬂavonoids and

their conjugates across a range of concentrations were studied by Noroozi et a1. (1998).
Their data suggested that the ﬂee form of ﬂavonoids was more protective against H202
induced DNA damage than their conjugated forms. Some polyphenolic compounds may
also exert an antioxidant effect by being involved in modulation of cellular functions
involving protein kinases such as kinase A, protein kinase C and phosphorylase kinase.
Caffeic acid has been shown to inhibit these kinases (Nardini et al., 2000).

Proaxidant character of polyphenols. Naturally occurring polyphenolic
compounds can act as pro-oxidants under appropriate conditions. This activity can
account for cytotoxic effects of these compounds observed against cancer cells (Hadi et
al., 2000). Previous studies ﬂom this lab have shown that antioxidants such as tannic
acid and ﬂavonoids either alone or in the presence of transition metal ions are capable of
generating oxygen radicals. In the presence of Cu (11), tannic acid causes DNA
degradation via hydroxyl radical production. It was concluded that that the structural
characteristics necessary for anti-oxidant effect, namely hydroxyl groups on the B ring of
this compound also contribute to its pro-oxidant activity (Khan et al., 2000). Beatty et al.
(2000) studied the effect of intake of ﬂavonols mainly quercetin, on oxidative damage in
36 healthy humans consuming either a low ﬂavonol‘ (LP) or a high ﬂavonol (HF) diet.
DNA damage products in leucocytes, plasma vitamin C and plasma quercetin
concentrations were measured. Although plasma quercetin concentrations were seen to
be signiﬁcantly higher in these subjects during the HF treatments, no changes were
observed in the either DNA base damage products or in plasma vitamin C, indicating that
dietary quercetin did not affect DNA damage in leukocytes. Yoshino and colleagues
(1999) demonstrated in vitro the potent cleavage of calf thymus and plasmid DNA by

myricetin, baicalein and quercetin. DNA treated with ﬂavonoids and copper produced 8-

10

OHdG, suggesting that reduction of cuprous ion by ﬂavonoids may contribute to DNA
cleavage and formation of base adducts. Quercetin was shown to signiﬁcantly increase
8-oxodG formation in the presence of copper in DNA ﬂagrnents ﬂom human p53 tumor
suppressor gene. Kaempferol and luteolin, which are structurally similar to quercetin,
increased production of this compound as well but to a lesser extent. Site speciﬁcity of
DNA damage was thymine and cytosine residues. These data support previous reports of
carcinogenicity of certain ﬂavonoids (Das et al., 1994; Sugimura et al., 1977; Sahu and
Washington, 1991 and Duthie et al., 1997) and also suggests that a DNA-copper-oxygen
complex rather than ﬂee hydroxyl radical might be involved in the DNA damage
(Y amashita et al., 1999). Sergediene et a1. (1999) demonstrated cytotoxicity of thirteen
compounds ﬂom the ﬂavonoids, gallic acid and caffeic acid groups of polyphenols.
These compounds showed a dose dependent cytotoxicity accompanied by
malondialdehyde production in promyelocytic lukemia cells (HL-60). This activity was
attributed to their pro-oxidant character.

Cardiovascular disease

There is abundant evidence that dietary polyphenols protect against LDL

oxidation in humans, a process implicated in the etiology of heart disease. Abu-Amsha
Caccetta et al. (2001) investigated the unique anti-atherosclerotic effect of red wine
consumption compared with other beverages. Their data demonstrated that de-
alcoholized red wine could reduce in viva lipid peroxidation. In a diet intervention study
involving adult human males, it was found that the oxidative stress associated with a high
fat diet could be effectively reduced by supplementation of red wine. In addition, the
total plasma antioxidant capacity was signiﬁcantly improved in the high fat group but not

to the extent that it was in the group that consumed a Mediterranean diet. Also, the high

11

level of DNA damage to blood leukocytes seen with the high fat diet was markedly
reduced with wine consumption (Leighton et al., 1999). Tewari and colleagues (2000)
studied the impact of additives in tea on the anti-oxidative properties of tea in healthy
human subjects. Serum lipid peroxidation was level was signiﬁcantly decreased thirty
minutes aﬁer ingestion of lemon tea and tea without milk, the effect of lemon tea being
greater than that of the latter. This result suggests that addition of lemon to tea increases
its antioxidant potential. Arts et a1 (2001) recently used data ﬂom the Zutphen Elderly
Study to evaluate the effect of a high catechin intake on incidence of stroke and ischemic
heart disease and mortality ﬂom these conditions. They found that catechin intake might
reduce the risk of mortality due to ischemic heart disease but not stroke irrespective of
the source of the compounds.

Importance of polyphenolic compounds in tea

Use and composition of tea. Use of tea ﬂom the plant Camellia sinensis
originated thousands of years ago in the orient and next to water is now the most widely
consumed beverage in the world. In ancient times this brew was used for therapeutic
purposes and owing to recent research uncovering a vast array of potentially beneﬁcial
properties, it comes as no surprise that tea is being touted as the new health drink
(Gultman and Ryu, 1996)

Brews ﬂom other plant sources are also. sometimes called ‘tea’ but generally, it is
the leaves of the plant Camellia sinensis that are considered to be real tea. The leaves
from this plant are processed differently to yield three types of tea namely black, green
and oolong tea. About 80 % of the tea produced worldwide is black tea and is consumed
primarily in the Western hemisphere. Enzymatic oxidation leading to condensation of

the polyphenols is the principle behind the manufacture of black tea. The condensation

12

products of this ‘fermentation’ step are the group of compounds known as thearubigens
and theaﬂavins, which are characteristic of black tea lending it its distinct color and
ﬂavor. Green tea represents 18 % of all tea produced and is popular in the East. Its
manufacture involves steaming or pan-ﬂying of ﬂesh leaves in order to prevent any
enzymatic oxidation of polyphenols ﬂom taking place. Only about 2% of all tea
produced is processed into oolong tea by partial fermentation of ﬂesh tea leaves. It is
consumed almost exclusively in Taiwan and China. The three types of tea differ in the
ratio of different tea polyphenols present in each. Polyphenols in tea are essentially the
catechins. Catechins comprise 30-42% of green tea on a dry weight basis. The four
major catechins in green tea are (—)-epigallocatechin gallate (EGCG), (-—)-epicatechin
(EC), (—)-epigallocatechin (EGC) and (—)-epicatechin gallate (ECG). Epigallocatechin
gallate is the largest ﬂaction contributing to about 50-80% of the total catechins present
in green tea. Catechins are characterized by di- or tri- hydroxyl group substitution of the
‘B’ ring and meta-5, 7-dihydroxy substitution of the ‘A’ ring as seen in Figure 1. It is
this structure that is known to chelate metal ions and prevent the formation of ﬂee
radicals. Tea also contains ﬂavonols quercetin, kaempferol and myricetin. In black tea,
the thearubigins and theaﬂavins account for 15 —20% and 2-6% respectively on a dry
weight basis. Tea leaves also contain about 2-5% caffeine and very small amounts of
theobromine and theophyline (Yang et al., 2002). The detailed composition of the
different types of tea is given in Table 1.

Anti- cancer effects of tea. Many studies have been published in recent years on
the potential anti-carcinogenic properties of tea and tea polyphenols. Tea polyphenol and
its various ﬂactions have been teSted for their antioxidant efﬁcacies, with green tea

having been more extensively studied than black tea (Mukhtar et al., 1999). The total

13

 

(-)-Epicatechin

OH

OH

OH OH

OH
OH

(-)-Epigallocatechin-3gallate

 

OH

Thearubigins
(R = Gallatc or other groups)

Figure 1 Structure of tea polyphenols

From Yang et a1. (2002)

OH

(-)-Epigallocatehcin

H
OH
OH OH
OH
OH

(-)-Epicatechin-3-gallate

    

 

0H
0 ..
H
OH
O
H .
OH
IR:

OH R1 R2
Thcaflavin OH OH
Theaflavin-B-gallate Gallate OH
'l‘heaﬂavin-3'-gallate OH Gallate
Theaﬂavin-3.3'-digallatc Gallatc Gallate

14

Table 1

Polyphenolic components of green and black teas as % dry weight of solids

 

 

Constituent Green tea Black tea

Flavanols 30-40 5-10
EGCG 10-15 4-5
ECG 3—10 3-4
EGC 3-10 1-2
EC 1-5 1-2

F lavandiols 2-3

Flavonols 5-10 6-8

Phenolic acids and depsides 3-5 10- 1 2

Theaﬂavins 3-6

Thearubigens 1 0-30

 

EGCG - epigallocatechin-3-gallate; ECG - epicatechin-3-gallate; EGC - epigallocatechin;
EC - epicatechin ' ‘

From Dreosti (1996)

15

antioxidant capacity of green and black tea is comparable to drinks made ﬂom other
ﬂuits and vegetables and may signiﬁcantly contribute to the intake of total daily
antioxidant capacity (Prior and Cao, 1999).

Epidemiological and clinical studies. Results ﬂom epidemiological studies to
evaluate the relationship between tea consumption and prevalence of cancer have been
inconclusive. Studies showing a negative association between tea consumption and risk
for developing cancer are ﬂom populations consuming very large quantities of the
beverage, namely Chinese and Japanese. Some studies that show no association or a
negative association between drinking tea and cancer are generally ﬂom the Western
countries. This result may be attributed to the fact that the amount of tea consumed is
minimal when compared to some eastern populations. A negative association between
green tea consumption and reduced risk for certain types of cancer has been demonstrated
in some studies but not in others. Gastric cancer risk was studied in a population-based
prospective cohort study in Japan. No association was noted between green tea
consumption and gastric cancer risk (Tsubono et al., 2001). Women consuming more
that 10 cups of green tea per day in Saitarna, Japan were likely to have a reduced risk for
cancer development at all sites combined as well as decreased risk for breast cancer
metastasis and recurrence (Irnai et al., 1997; Nakachi et al., 1998). In the NHANES I
Follow Up Study a general inverse association was observed between tea consumption of
' 2 1.5 cups/day and colon cancer in males and females in the United States (Su and Arab,
2000). Recently in a large population-based prospective cohort study in Sweden
(Swedish Mammography Screening Cohort), association between consumption of tea,
coffee and caffeine and risk of breast cancer was investigated. No association was found

between the caffeine containing beverages and caffeine and incidence of the disease.

16

This result was observed in women consuming either 1 cup of tea or less per week or 2-3
cups or more per day (Michels et al., 2002). Vermeer et al. (1999) investigated the
effects of ascorbic acid and green tea separately on the formation of N-nitrosodimethyl
amine (NDMA) and N-nitrosopiperadine formation in 25 healthy females. While there
was a decrease in urinary NDMA excretion with ascorbic acid and 4 cups of green tea per
day during days 4-7 of the study, there was actually a signiﬁcant increase in NDMA
excretion with consumption of 8 cups of green tea per day during this time. These
conﬂicting results indicate the difﬁculty in making generalized recommendations about
increasing tea consumption and the need for more studies in human populations to
determine the true nature of protection, if any, offered by tea against cancer.

In vitro and animal studies. Examples of recent research in this area include the
paper by Steele et al. (2000) in which extracts of both black tea and green tea were
effective as chemopreventive agents when tested with nine standard cells culture assays.
Results provided strong evidence that these two extracts were anti-mutagenic, anti-
proliferative and anti-neoplastic. Cell lines included mouse mammary organ cultures, rat
tracheal epithelial cells and human lung tumor epithelial cells. Jurkat T cells treated with
green tea extract at 10 mg/L in medium showed a marked decrease in F e+2 induced
oxidative damage as evidenced by a reduction in malondialdehyde production and DNA
damage (Erba at al., 1999). The effect was attributed to the epigallocatechin gallate
component of green tea and TNF-alpha release. The chemopreventive beneﬁt of black,
and green tea extracts and puriﬁed polyphenolic components against pancreatic and
prostate cancers was examined in vitro by Lyn-Cook et a1. (1999). Polyphenolic
components included a mixture of green tea polyphenols (GTP) and mixtures of black tea

polyphenols and theaﬂavins (BTP and MF) and puriﬁed polyphenols epicatechin-3-

17

gallate (ECG) and epigallocatechin-3-gallate (EGCG). The tea extracts, as well as the
ECG and EGCG, inhibited growth of human pancreatic adenocarcinoma and prostrate
tumor cell lines and also decreased the expression of the k—ras gene. A decrease in
expression of mdr-l gene was seen only with the two tea extracts. This result suggests a
chemopreventive effect against pancreatic and prostate cancer by way of modulating
expression of relevant genes. A study by Kennedy et al. (1999) identiﬁed thiol groups as
a novel target for the toxicity of green tea polyphenols in Ehrlich Ascites tumor cells and
a regulatory role for green tea by reducing SH groups in tumor inhibition. A recent study
by F eng et al. (2002) evaluated the antioxidant effects of black tea polyphenols, namely
theaﬂavins (theaﬂavin, theaﬂavin monogallate and theaﬂavin digallate). These
compounds showed inhibitory effects against H202 and tert-butyl hyroperoxide induced
cytotoxicity, cellular oxidative stress and DNA damage in RL-34 cell line. Their results
demonstrated that theaﬂavins could prevent oxidative DNA damage in cell cultures by
suppressing cytochrome P450 1A1, a metabolic activator of certain chemical
carcinogens.

Anti-angiogenic effects, of catechins. Jung et a1. (2001) investigated the effect of
green tea catechins on intracellular signaling and induction of Vascular Endothelial
Growth Factor (VEGF) in human colon cancer cells, which is of importance due to
cancer being an angiogenesis-dependent disease. This study with HT29 human colon
cancer cell line showed that EGCG had a dose dependent inhibitory effect on Erk-l and
Erk-2 activation. No effect was seen with other catechins.. BALB/c nude mice
inoculated with HT29 cells (in vivo study) and green tea catechins for 20 days showed
(1 .Smg/d) 58% reduction in tumor growth, decrease in microvessel density (30%) and

tumor proliferation (27%). EC, ECG, EGC and EGCG inhibited growth, migration and

18

tube formation, which are models of angiogenesis, in human umbilical vein endothelial
cells. EGCG alone was also found to reduce binding of VEGF to its receptor thereby
affecting downstream signaling (Kondo et al., 2002). It has also been suggested in the
same model that EGCG acts as an angiogenesis inhibitor by modulating protease activity
during endothelial morphogenesis (Singh et al., 2002). In a recently published study by
Masuda et al. (2002) EGCG was found to inhibit VEGF production in head and neck
squamous carcinoma (Y CU-H891 HNSCC) and breast carcinoma (WA-MB-23l) cell
lines. Therefore EGCG may used to prevent these two types of cancer by anti-angiogenic
activity.

Eﬂects of food matrix. A recent study by Km] and colleagues (2001) aimed to
evaluate the effect of a food matrix on green and black tea absorption using a novel in
vitro gastrointestinal model. In addition to investigating absorption of tea polyphenols,
they also studied the effectiveness of the tea extracts against mutagenicity induced by the
mutagen 2-amino-3, 8-dimethylimidazo[4,5-f] quinoxaline (MeIQx) after undergoing
digestion. Addition of milk or a homogenized standard breakfast to tea was evaluated.
Whole milk, partially skimmed milk and skim milk were used in this experiment and the
breakfast consisted of brown and white bread, cheese, butter, marmalade and water. The
tea dialysate obtained ﬂom the jejunal compartment of the model inhibited mutagenicity
of the mutagen MeIQx. Addition of milk to the tea during digestion partially decreased
the anti-mutagenic effect of both teas, while introduction of the homogenized breakfast
completely abolished the effect. This reduction in anti-mutagenicity was proportional to
a decrease in the polyphenol content of the dialysates. These results are in agreement
with other studies (Arts et al., 2002, Langley-Evans, 2000), which show an inhibitory

effect of milk on the antioxidant activity of tea both in vitro and in humans. However,

19

some other reports do not support these ﬁndings (Richelle et al., 2001 , Leenen et al.,
2000)

The vast amounts of data that have been generated ﬂom investigations on tea
reveal that although there may be a protective role for this beverage or its active
principles against cancer, evidence is not consistent. In addition to being inconclusive,
the effect of physiological concentrations of tea and the effect of other foods on the
various putative mechanisms of protection by tea are very limited. Therefore well-

planned studies need to be conducted in this area to further already present ﬁndings.

20

JUSTIFICATION AND OBJECTIVES
Experimental evidence indicates that tea and its polyphenols are anti-
carcinogenic. However, most of the promising results are ﬂom studies that used tea as the
sole food source or isolated compounds of interest ﬂom tea at concentrations greater than
what would be seen physiologically. Data are limited and unclear on the observed
beneﬁcial effects being preserved when these compounds are consumed in their naturally
occurring state and more importantly as part of a composite meal. The present study will
help to elucidate the effect of digestion and meal components on the absorption of
polyphenols and antioxidant effect of green and black tea.
The principal objective of this research was to study the effect of tea decoction on
hydrogen peroxide mediated DNA damage in CacoZ cells. Speciﬁc objectives were,
1. To determine the inﬂuence of simulated digestive process on potential
cytoprotective effect of tea in Cac02 cells.
2. To determine the effect of the presence of high protein and high iron foods
during simulated digestion on the potential cytoprotective property of tea.
In order to study the effect of digestion of green and black tea in the milieu of a food
matrix on their potential antioxidant activity the ﬁrst hypothesis to be tested was

Null hypothesis 1

Simulated digestion has no inﬂuence on the potential cytoprotective effect
of tea against oxidative damage by H202 in Cac02 cells

Null hypothesis 2

Combining high protein and high iron food with tea during digestion will
not decrease the cytoprotective effect of tea against oxidative damage by

H202 in Cac02 cells

21

MATERIALS AND METHODS

Research design

This study was designed to compare the antioxidant effect of digested and

undigested tea on colon cells. The effects of iron and protein on the anti-oxidative effect

of tea were determined as well. “Test meals” formulated with black tea or green tea

decoctions were subjected to a simulated digestive process either alone or in the presence

of a high iron cereal or milk. The effects of these test meals were compared with tea

decoction that had not undergone in vitro digestion. Antioxidant effect was evaluated in

Cac02 colon cancer cell line. The cells were pre-treated with the test meals and

subsequently subjected to hydrogen peroxide induced oxidative stress. Resultant damage

to cells was assessed in terms of single strand breaks and formation of oxidized bases in

DNA.

Materials

Tea

Cereal

Milk

Water

Glassware

Lipton Yellow Label Tea (blended granular assam tea) and Lung Ching
China Green Tea (green leaf tea) imported ﬂom India and Hong Kong,
respectively were purchased ﬂom local stores. ’

Product 19 (Kellogg Company, MI) was selected because one 30 g serving
of the breakfast cereal provided 100% of the Daily Value of iron (18 mg).
(Table 2)

2% milk was purchased locally (Country Fresh, Grand Rapids, MI). Milk
used in all the experiments was ﬂom the same 1 pint container (Table 2)
Double deionized (DD) water was used in all in vitro digestion
experiments and Nanopure® water was used for DNA digestion.

Acid washed glassware was used in all the experiments.

22

Table 2

Nutrient content of 2% milk and Product l9® breakfast cereal 1

 

 

2 % Milk Kellogg Product l9®
(240 ml/l cup) (30 g)
Kcals 130 100
Total fat (g) 5 0
Total carbohydrate (g) 13 25
Protein (g) 8 2
Iron (mg) 1 " 0 18 (5100% DV)

 

" 1 Label information On milk bottle and cereal box.

23

Chemicals

Pepsin ﬂom porcine stomach mucosa (2500 U/mg protein), pancreatin (ﬂom
porcine pancreas), porcine bile extract (glycine and taurine conjugates of hyodeoxycholic
acid) and sodium bicarbonate were purchased ﬂom Sigma Chemical Co. (St. Louis, MO).
Hydrogen peroxide and sodium hydroxide were ﬂom J .T. Baker (Phillipsburg, NJ).
Sodium chloride was purchased ﬂom EM SCIENCE (Gibbstown, NJ). Potassium
chloride and potassium phosphate were obtained ﬂom Mallinckrodt (Paris, KY) and
sodium phosphate ﬂom MCB (Norwood, OH). Sodium ethylenediamine-tetraacetic acid
(Na-EDTA) and TRIS were purchased ﬂom Gibco BRL (Grand Island, NY). Triton X-
100 was obtained ﬂom Research Products International Corp. (Elk Grove Village, IL)

The following cell culture materials were form Gibco BRL: Dulbecco’s Modiﬁed
Eagle’s Medium (DMEM: cat. no. 23700-024) containing non-essential amino acids, L-
glutamine, pyridoxine hydrochloride, 25 mmol/L N-[2-Hydroxyethyl]piperazine-N'-[2-
ethanesulfonic acid] (HEPES) and a high concentration of glucose (4500 mg/L) which
was used to culture cells; fetal bovine serum (FBS) and Minimtun Essential Medium
(MEM). The MEM was supplemented with 10 mmol/L piperazine-N, N'- bis-[2-
ethanesulfonic acid] (PIPES), 1 % antibiotic-antimycotic solution, hydrocortisone
(4 mg/L), insulin (5 mg/L), selenium (5 jig/L) and 34 ug/L triiodothyronine
(all purchased ﬂom Sigma Chemical Co.), insulin (5 mg/L) and epidermal growth factor
(20 pg/L) ﬂom Gibco BRL. Trypsin-EDTA (25%) was purchased ﬂom Sigma- Aldrich
(St. Louis, MO).

The DNA isolation kit was purchased ﬂom CPG Biotech (Lincoln Park, NJ).

Chemicals used for DNA digestion included: DNase I and Nuclease PI purchased ﬂom

24

Roche Diagnostics Corporation, (Indianapolis, IN), Exonuclease III ﬂom USB
Corporation, (Cleveland, OH) and Bacterial Alkaline Phosphatase (from E. coli C75)
Amersham Pharmacia Biotech Inc., (Piscataway, NJ). The Highly Sensitive Kit for
measurement of 8-hydroxy-2-’deoxyguanosine was purchased ﬂom Genox Corporation
(Baltimore, MD).
Cell culture

CacoZ cells are derived ﬂom human colon cancer cells and are extensively used
in in vitro studies in which a representation of the intestinal epithelium is required. This
cell line has been shown to transform into intestinal cells when cultured and demonstrates
the same morphological and biochemical characteristics as mature enterocytes (Pinto et
al., 1983). Therefore, the Cac02 cell line has been used as a model to study a variety of
intestinal functions involved in uptake and transport of nutrients such as minerals and
amino acids (Han et al., 1994). Halliwell et al. (2000) have suggested the importance of
anti-carcinogenic activity of dietary compounds in the gut. In the present study Cac02
cells were used tostudy possible cellular protection offered by tea against oxidative
damage.

Cac02 cells, an anchorage dependent cell line, were purchased ﬂom American
Type Culture Collection (Rockville, MD). The cells were maintained in Dulbecco’s
Modiﬁed Eagle’s Medium (DMEM) with 10% (v/v) fetal bovine serum and 1%
antibiotic-antimycotic solution (Gibco BRL) in 75cm2 ﬂasks (Costar, Cambridge, MA) at
37 °C in an incubator.

Cells were used between passages 25-33 and seeded at a density of 50,000
cells/cm2 in collagen coated 6-well plates (Costar). Media was changed every second day

up until the day of the experiment.

25

Cell Viability

Viability of cells subjected to hydrogen peroxide treatment was determined using
Trypan blue. Cells were harvested with media at 10,000 cells /ml. An aliquot (20 pl) of
the cell suspension was mixed with equal volume of trypan blue, loaded onto a
hemocytometer and viewed under 40X magniﬁcation. Cells that were dying or dead
absorbed the dye and were stained a dark blue whereas live cells appeared pale blue. The
number of dead cells as well as live cells was counted to yield percent viability of Cac02
cells.
H202 induced oxidation: preliminary experiments

Preliminary experiments were done to determine concentrations of hydrogen
peroxide required to produce oxidative damage. Cac02 cell monolayers grown in 6-well
culture plates were rinsed twice with PBS and treated with either 200 or 500 uM H202 in
PBS on ice for 10 and 20 min. Following the incubation period, cells were rinsed twice
with PBS and harvested with trypsin - EDTA for measurement of DNA damage. Cells
incubated with 500 uM H202 showed the greatest evidence of oxidative damage based on
number and tail length of comets determined by alkaline single cell gel electrophoresis.
Hence this concentration with 20-minute incubation period was selected for all further
experiments.
Simulated digestion

In vitro digestion of samples was conducted by the method of Au and Reddy
(2000). The digestive process involved a gastric and an intestinal phase. A 4 g sample of
black tea or green tea was steeped in 50 ml boiling water for 5 min. The infusions were
strained and a10 ml sample of tea decoction + 1.2 g of cereal or 1 ml milk was used in

each experiment. These speciﬁc amounts of tea, cereal and milk were proportional to a

26

meal comprising a serving of double strength tea with 25 g of milk added to it and 30 g
serving of cereal. The pH of the sample was adjusted to 2 with 0.1N HCl. A 0.5 ml
aliquot of ﬂeshly prepared pepsin (0.08g/m1 0.1mol/L HCl) was added to the sample and
incubated in a 37°C shaking water bath (Dubnoff metabolic shaking incubator,
GCA/Precision Scientiﬁc, Chicago, IL.) for 1 h at a speed of 90 rpm. The pH of the
sample was then adjusted to 6 with drop-wise addition of 0. lmol/L NaHCO3 solution. A
2.5 ml aliquot of pancreatin-bile solution (0.12 g bile extract and 0.02 g pancreatin in 5
ml 0.1mol/L NaHCOg) was then added to the sample and pH adjusted to 7 with 0.1N
NaOH. The sample was then incubated at 37°C in a shaking water bath for 30 min.
Volume of the sample was brought up to 25 ml with 120 mmol NaCl-S mmol KCl
solution. For the digest blank, DD water replaced the tea decoction. After centrifugation
for 30 min at 6500 rpm at 4°C, supematants were collected and reﬂigerated overnight
(4°C). Aliquots (1.5 m1) of supernatant ﬂom each sample were taken and used to pre-
treat Cac02 cells in subsequent antioxidant studies.
Treatment of cells

Cac02 cells were seeded at a density of 25,000cells/ ml (1,000,000 cells/well) in
6-well cell culture plates for Comet assay and measurement of 8-OHdG. Cell
monolayers were maintained in DMEM at 37°C in a humidiﬁed incubator with 95% air
and 5% CO2 environment. Media was changed on altemate days. Cells were used
between days 6-8 post-seeding when the monolayers had reached conﬂuence.

Prior to treatment with aliquots of treatment samples, cell layers were washed
twice with MEM at 37°C. An aliquot (1 m1) of MEM was placed in each well to which

1.5 ml of digest was added. Freshly brewed tea decoction made up to the same volume

27

and pH as the digested tea samples served as ‘undigested’ tea test meal. The cells were
allowed to incubate at 37°C for an hour. At the end of this period, cell layers were
washed twice with phosphate buffered saline (PBS) and 2 ml of 500uM H202 in PBS
placed in each well. The plates were placed on ice for 20 min. At the end of the
incubation, cells were washed twice with PBS and 0.5 ml of trypsin-EDTA placed on
each monolayer. The trypsin was withdrawn after 30 seconds and the plates incubated
for 5 min at 37 °C. Cells were then harvested in cold media, centriﬁrged and pellets used
directly either for comet assay or for DNA isolation.

For the experiments comparing tea and tea digests, 1.5 ml aliquots of each of the
four samples (undigested tea, digested tea, digested tea + cereal, digested tea + milk) and
the experimental blank were placed in 2 wells each of a 6 well plate. Two additional
wells were treated with H202 alone and two received no treatment (control). The amount
of MEM added to-these wells was 2.5 ml to give same total volume as in the digest
treated wells. A total of 14 wells were used for each replicate of the experiment.

Each experiment was conducted in triplicate for green and for black tea. DNA
was extracted ﬂom cells ﬂom each well and subjected to digestion. Each sample of
digested DNA was then used in duplicate for measurement of 8-0HdG with ELISA.

A ﬂow chart of the experimental design is shown in Figure 2.

28

Tea infusion (10 ml) g Tea infusion (10 m1) g Tea infusion (10 ml)
+ cereal (1.2 g) + milk (1 g)

/

Adjust pH to 2 with BC]
I
Addition of 0.5mL pepsin
l
Gastric digestion, 1h at 37°C
l
Adjust pH to 6 with NaHCO3
1
Addition of 2.5 m1 pancreatin-bile
I
Adjust pH to 7
l
Intestinal digestion, 30min at 37°C
l
Adjust volume to 25ml with NaCl-KCl solution
l
Centrifuge digest for 30 min at 6500 rpm
l

Aliquot of supernatant placed on
Cac02 cell monolayers

l
Incubate at 37°C for 1h
l
Rinse cells with phosphate buffered saline (PBS)
l
Treat cells with 500 M H202

on ice for 20 min

_ i
Rinse cells with PBS

l
Harvest cells with trypsin-EDTA for measurement of DNA damage

Figure 2 Flow diagram of experimental design

29

Estimation of DNA damage by Alkaline Single Cell Gel Electrophoresis (Comet
Assay)

Oxidative DNA damage was assessed by employing the Comet assay. This
method is an alkaline modiﬁcation of the micro gel electrophoresis technique used to
detect single stranded DNA breaks and alkali—labile sites (Singh et al., 1988). Cells are
embedded in agarose gel on microscope slides, lysed by detergents and high salt, and
then electrophoresed for a short period under alkaline conditions. During electrophoresis,
the high molecular weight DNA partly migrates towards the anode. When viewed under
magniﬁcation, the migrated DNA appears to be the ‘tail’ and the remainder that has not
moved forms the ‘center’ of the comet. These ‘comets’ are quantiﬁed by staining with
ethidium bromide (2 jig/ml) and measuring the tail length of comets.

Cac02 cells pre-treated with test meals and treated with H202 were harvested and
suspended in 75 ul of 0.5 % Low Melting Point (LMP) agarose (Sigma-Aldrich Co., St.
Louis, MO). This agarose was pipetted onto a glass slide precoated with the same
amount and concentration of LMP agarose. The agarose was set for 10 min at 4°C. A
third layer of the agarose was pipetted onto the slide and set similarly. The cells were
lysed for 1 h in a lysing solution (2.5 M NaCl, 100 mM Na2—EDTA, 10 mM Tris and 1%
Triton X-100) at pH 10 and 4°C. The slides were then placed in a horizontal
electrophoresis unit (BRL, Life Technologies, Inc., Gaithersburg, MD) containing
electrophoresis buffer (lmM Na2—EDTA and 300 mM NaOH) at pH 12.7 and 4°C for 40
min to allow DNA unwinding. Electrophoresiswas canied out at 25 V for 30 min (Bio-
Rad 200/2.0-power supply, Hercules, CA). The slides were then washed three times with

a washing solution (0.4 M Tris) at pH 7.5, stained with ethidium bromide (2 jig/ml) and

30

viewed under a ﬂuorescence microscope (NIKON Labophot, Japan) connected to a
Kodak Microscopy Documentation System (MDS) 290, at 10X magniﬁcation to be
scored visually. A total of 50 cells were counted whenever possible. Cells with damage
were identiﬁed to obtain the percentage of comets formed. The tail lengths of 50 comets
counted sequentially were measured using NIH Image (1.62) software.

Estimation of DNA damage by measurement of 8-Hydroxy-2'-deoxyguanosine

(8-OHdG)

DNA isolation. CacoZ cell monolayers were pre-incubated with test meals and
treated with 500 uM H202 for 20 min on ice. Cells were then washed twice with PBS,
trypsinized with 0.5 ml trypsin-EDTA (which was removed after 30 seconds) and
incubated for 5 min at 37°C. Cold media (1 ml) was then added to each well. The cell
layers were harvested and centrifuged for 2 min at 5000 rpm. Resultant cell pellets were
then used directly for DNA extraction with the DNA isolation kit. Isolated DNA was
stored at 4°C until further use.

DNA digestion. Single stranded DNA was obtained by incubating 10 pg DNA
with 180 units of Exonuclease III at 37°C for 1 h, heating at 95°C for 5 min and followed
by rapid chilling on ice. Subsequent digestion of DNA to nucleosides was done by
incubation with 0.6 units Nuclease P1 for 1 h at 37°C and another hour at 37°C with 0.6
units bacterial alkaline phosphatase. The digest was used to measure 8-OHdG.
Randomly selected DNA samples (digested) were subjected to electrophoresis on a 1 %

agarose gel to conﬁrm digestion. The appearance of smears instead of bands on the gel

showed that the DNA had been digested (Figure 3).

31

,v
tl

M 1234 5678

“NW li‘(‘," ”WW ‘mg‘ml

 

Figure 3 Electrophoresis of DNA and digested DNA of Cac02 cells on a 1% agarose gel. DNA isolated
from Cac02 cells was subjected to digestion. Digested and undigested DNA were then electrophoresed to
conﬁrm digestion. The appearance of smears instead of bands indicates that DNA was digested. Lanes on
the gel indicated by letters and numbers ﬂom left to right are:
M Molecular weight marker
1 Green tea followed by hydrogen peroxide treatment
2 No treatment
3,4 500uM H202
5,6,7,8 Digested DNA of 1,2, 3 and 4
* 1.6 Kb band
# 1 Kb band

32

8-0HdG Assay. 8-hydroxy-2'-deoxyguanosine formation was measured
according to the protocol of the competitive ELISA kit (Toyokuni et al., 1997). Digested
DNA (50 pL) of various samples was added to the wells of a microtitre plate pre-coated
with 8-OHdG. Primary antibody N45.l (50 uL) was then added and the plate allowed to
incubate overnight at 4°C. The plate was then washed three times with the washing
solution (PBS containing 1% BSA and 0.5 % Tween 20). Secondary antibody
peroxidase-conjugated anti-mouse IgG antiserum (100 uL) was then added to each well
and allowed to incubate at room temperature for l h. Washing was repeated and 100 pL
of a chromatic solution (citrate buffer, pH 5, containing 0.4 mg/ml of o-
phenylenediamine and 0.015% H202) was added to each well. The plate was shaken
continuously for 15 min in the dark. The reaction was stopped by addition of 100 uL of a
reaction terminating solution (2M sulfuric acid). Absorbance readings were taken at 450
nm using a Kinetic ‘Microplate Reader (Molecular Devices Corporation, Sunnyvale, CA)
and concentration of 8-OHdG of unknowns was calculated ﬂom the standard curve
plotted on semi-log scale using Microsoft Excel (Figure 4). The standards ranged ﬂom

0.125 ng/ml to 10 ng/ml and results were expressed in terms of ng/mg DNA.

33

 

 

 

 

 

CD. at 450 nm

 

 

 

 

8-OHdG (nglml)

Figure 4 Standard curve of 8-hydroxy-2'-deoxyguanosine (8-OHdG) by ELISA plotted on a semi-
log scale. Standards used were 0.125 ng/ml, 0.25 ng/ml, 0.5 ng/ml, 1 ng/ml, 4 ng/ml and 10 ng/ml.
Equation for logarithmic trendline (dotted line): y = -0.2206Ln(x) + 0.9921 and R2 = 0.99

34

Statistical Analysis

One way analysis of variance (AN OVA) was employed as the statistical tool to
detect signiﬁcant differences between various groups: untreated cells, cells treated with
hydrogen peroxide alone, cells pre-treated with tea decoction, digested tea decoction and
tea digested with cereal and with milk. Results ﬂom control, hydrogen peroxide and
blank digest treatment groups for black tea and green tea were pooled together before
applying the analysis of variance. Fisher’s LSD was the post-hoe test employed to
identify which groups were signiﬁcantly different ﬂom each other at p_<_ 0.05. For results
ﬂom the Comet assay, AN OVA and Fisher’s LSD were employed to see if there were
signiﬁcant differences between control cells (no treatment), cells treated with hydrogen

peroxide and cells pre-treated with green tea.

35

RESULTS

This study was conducted to evaluate the effect of the digestive process on the
antioxidant effect of green and black tea on Cac02 cells. The possibility that high iron or
protein might inﬂuence the anti-oxidative effect of tea also was studied. The different
‘test meals’ selected were plain tea decoction (boiling water infusion), tea decoction that
underwent in vitro digestion either by itself or in combination with a high iron cereal or
partially skimmed milk. Hydrogen peroxide was selected as the oxidizing agent to induce

DNA damage. Damage to DNA in colon cells was evaluated as single strand breaks or as

production of 8- hydroxy-2'—deoxyguanosine.

Effect of hydrogen peroxide on viability of Cac02 cells and formation of DNA single

strand breaks

For the purpose of testing the effect of H202 on viability of CacoZ cells, an
experiment was done in which cells were seeded at 2x106 cells/ml into 6-well plates.
When the monolayers had reached 90% conﬂuence, layers were rinsed twice with PBS
and 2 ml of either 200 uM or 500 uM H202 in PBS was placed in wells on ice for 10, 20
and 40 minutes. PBS with no H202 served as control. Viability of cells was tested at the
different time points for the two concentrations and control. Cells were stained with
trypan blue and viewed under magniﬁcation. Cells that were dead appeared dark blue
under magniﬁcation and live cells were stained pale blue. Viability of cells was greater

than 90 % and was not different between the two treatments and at the three time points.

36

Effect of H202 on DNA single strand breaks in Cac02 cells

Results for formation of single strand breaks in DNA in Cac02 cells are given in
Figure 5. Cells were treated with 200 and 500 uM H202 for 20 min on ice. The
percentage of comets formed with 200 uM H202 was 26 % and that with 500 pM was
51.5 % when compared to controls (untreated cells). Tail lengths of the comets for cells
treated with 200 and 500 uM H202 were 694 and 872 arbitrary units, respectively. Based
on these results, the 500 uM H202 treatment for 20 minutes was selected for application
in the Comet assay. Figure 6 shows a comparison of tail lengths found in a typical

experiment.
Effect of green tea pre-treatment on DNA single strand breaks in Cac02 cells

Pre-treatment of Cac02 cells with green tea infusion for 2 h was effective in
protecting against H202 induced DNA single strand breaks. Figures 5 and 6 illustrate
typical result of the comet assay for cells treated with H202 with and without pre-
treatment with green tea. The percentage of comets produced when pre-treated with
green tea was 36 i 12 %, which was signiﬁcantly lower when compared to hydrogen

peroxide treatment alone. 0

Further experiments with this method were abandoned due to lack of
reproducibility in results. Treatment of cells with green tea caused agglutination of cells
during harvesting. This inability to obtain a single cell suspension in LMP agarose
essential for even distribution of cells on slides resulted in densely packed cells making

for difﬁculty in counting. It was therefore concluded that this method was unsuitable for

37

 

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Treatments

Figure 5 Effect of hydrogen peroxide and green tea treatment on percentage of comets formed in Cac02
cells. Data are mean i SD, N=5. Cells were treated with 200 or 500 M H202 for 20 min on ice or
pretreated with green tea decoction prior to 500uM H202 treatment. Bars sharing a letter are signiﬁcantly
different at p S 0.05. H - hydrogen peroxide; Control - untreated cells;

38

 

Figure 6 Effect of hydrogen peroxide and green tea on tail length of comets formed in Cac02 cells in a
typical experiment. Cells were treated with 500 11M H202 for 20 min on ice or pretreated with green tea
decoction prior to 500 uM H202 treatment. (a) No H202; (b) 500 uM H202; (c) Green tea decoction
followed by 500 uM H 202, Images in this thesis are presented in color.

39

the model used in this study. However, these results indicating substantial levels of DNA
damage in Caco-2 cells with 500 pM H202 were used as a basis for the concentration of
H202 to be used in an alternative method to estimate DNA damage, namely measurement

of 8-0HdG formation.
Production of 8-hydroxy-2'-deoxyguanosine (8-OHdG)

Eﬂect of 5 00 MW H 202. Data on the effect of 500uM H202 on 8-0HdG
production in Cac02 cells are given in Tables 3 and 4. Concentration of 8-OHdG in cells
treated with hydrogen peroxide was similar to that in untreated cells (control), 0.291 i
0.047 ng/ug DNA and 0.285 i 0.031, respectively. Cells bathed in MEM for the duration
of the experiment until harvesting for DNA extraction served as control. In contrast to
evidence of DNA damage in the comet assay, this concentration of the hydrogen peroxide

resulted in little damage in the form of adduct formation of guanosine.

Effect of pre-treatment with green tea. The 8-OHdG concentration of cells pre-
treated with green tea infusion prior to exposure to the oxidizing agent was 0.308i0.043
ng/ug DNA (Table 3). After undergoing simulated peptic and intestinal digestion, the
same concentration of tea induced a similar production of 8-OHdG (0.306 t 0.089 ng/ltg
DNA). Although! cells treated with the blank digest, which contained digestive enzymes
but no tea had somewhat lower concentration of 8-OHdG than the peroxide treated cells,
the difference was not signiﬁcant. When a dietary source of high iron was added to tea,
there was a non-signiﬁcant increase in production of the toxic compound,

0.344 t. 0.088 ng 8—OHdG /ug DNA, which corresponded to an increase of 18%

compared to hydrogen peroxide treatment. Addition of milk to the tea decoction

40

Table 3

Concentration of 8-hydroxy—2'—deoxyguanosine (8-OHdG) in Cac02 cells pre-treated
with green tea decoction, digested tea decoction, digested tea plus milk or cereal

followed by treatment with 500 uM hydrogen peroxide (H202) 1' 2

 

 

Sample 8-OHdG (ng/ug DNA)
Control (untreated cells) 0.285 i 0.031
Oxidizing agent (500 uM H202) 0.291 i 0.047

Green tea infusions
Undigested 0.308 2‘. 0.043

In vitro digests

No tea (blank) 3 0.254 a 0.045
Tea 0.306 a 0.089
Tea + cereal 0.344 :I: 0.088
Tea +milk 0.360 a 0.071‘ 4

 

1 Data are mean i SD, N=6 except for control, cells treated with H202 and cells pre-treated with blank
digest for which N=10 "
2 Cells were treated with 500 uM H202 for 20 min after 1 h incubation with green tea decoction,
digested tea decoction or tea decoctiOn digested With cereal or milk.
3 DD water replaced tea in the digest blank.
4 "' Signiﬁcantly different ﬂom 500 uM H202 at ps 0.05

41

Table 4

Concentration of 8-hydroxy-2’-deoxyguanosine (8-OHdG) in Cac02 cells pre-treated
with black tea decoction, digested tea decoction, digested tea plus milk or cereal
followed by treatment with 500 uM hydrogen peroxide 01202) 1’ 2

 

 

Sample 8-OHdG (ng/ug DNA)
Control (untreated cells) 0.285 i 0.031
Oxidizing agent (500 uM H202) 0.291 i 0.047

Black tea infusions

Undigested 0.313 t 0.030

In vitro digests

No tea (blank) 3 0.254 a 0.045
Tea 0.316 a 0.030
Tea + cereal 0.282 t 0.037
Tea + milk . 0.295 a 0.027

 

1 Data are mean i SD, N=6 except for control, cells treated with H202 and cells pre-treated with blank
digest for which N=10

2 Cells were treated with 500 uM H202 for 20 min after 1 h incubationwith black tea decoction,
digested tea decoction or tea decoction digested with cereal or milk.

3 DD water replaced tea in the digest blank.

42

followed by in vitro digestion caused a signiﬁcant increase in generation of the oxidized
base (0.360 :t 0.071 ng 8-OHdG /p.g DNA), which represented a change of 24%
compared to hydrogen peroxide treatment alone. This result suggests a possible

pro-oxidant effect of milk combined with tea.

Effect of pre-treatment with black tea. Results for the effect of black tea
treatment on 8-OHdG production are summarized in Table 4. Colon cancer cells treated
with a boiling water infusion of black tea had 0.313 i 0.030 ng/ug DNA of the oxidized
product, which was not statistically different ﬂom hydrogen peroxide treated cells. A
similar concentration of 8-OHdG (0.316: 0.030 ng/pg DNA) was formed when the same
infusion was subjected to in vitro digestion before ﬂeeting the cells. This result was
similar to that for green tea and indicates that the digestive process used in this model
does not inﬂuence the chemical characteristics of tea. Addition of a high iron breakfast
cereal to the tea during the digestive process caused a negligible reduction in 8-OHdG
formation (0.282 t 0.037 rig/pg DNA) compared to cells treated with H202 alone.
Similarly, the presence of milk also caused a reduction in generation of this compound
(0.295 .+. 0.027 ng/ug DNA), but the decrease was somewhat less than that for cereal.

Figure 7 summarizes results for both green and black tea. It can be seen that cells
treated with green tea plus cereal had a signiﬁcantly higher content of 8-OHdG compared
with untreated cells and cells ﬂom the digest blank group. The addition of milk to green
tea during digestion resulted in signiﬁcantly higher DNA damage in Cac02 cells that
were pre-treated with this digest in comparison to untreated cells, hydrogen peroxide

treated cells and cells treated with the digest blank.

43

 

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Figure 7 8-OHdG content in Cac02 cells pre-treated with green or black tea decoction (4 g tea leaves in 50
ml boiling water) or tea digests, followed by hydrogen peroxide treatment (500 M for 20 min). Black and
green tea digests were prepared by in vitro digestion of tea decoction either by itself or with cereal or milk.
Bars sharing a common letter are signiﬁcantly different at p S 0.05 with the exception of GTC and GTM,
and BT and BTD, which are not signiﬁcantly different ﬂom each other.

C - control; H - 500uM hydrogen peroxide; B — digest blank; GT - green tea decoction; BT - black tea
decoction; GTD - digested green tea; BTD - digested black tea; GTC - green tea + cereal; BTC - black tea +
cereal; GTM - green tea + milk; BTM - black tea + milk

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44

Comparisons made between green and black tea groups showed that 8-OHdG
level in cells pre-treated with green tea digested with milk was signiﬁcantly higher
compared to black tea with milk. Similarly, the addition of cereal to green tea resulted in
signiﬁcantly greater DNA damage in cells compared to pre-treatment with black tea and

cereal.

45

DISCUSSION

Reactive oxygen species may be a major contributor to development of chronic
diseases such as cancer and heart disease (Halliwell et al., 2000). Epidemiological
studies strongly point to a beneﬁcial role for antioxidants in prevention of these diseases.
Antioxidant vitamins C, E and carotenoids are found in abundance in ﬁ'uits and
vegetables. However, this food group is also a major source of plant polyphenols,
‘non-nutrient’ compounds known to possess strong anti-oxidative characteristics.
Because of the high polyphenolic content in green and black tea, there is also
considerable interest in the antioxidant/anticancer activity of green and black tea.
Catechins, a sub group of the group ﬂavonoids are the major polyphenolic compounds in
tea. Numerous studies have demonstrated a protective role for these beverages against
oxidation related diseases such as cardiovascular disease and cancer. However there has
been a lack of focus on these same properties in the milieu of a ‘meal’ wherein other

nutrients are present and may interact in a manner, which modiﬁes chronic disease risk.

Tea decoctions, the form in which tea is normally consumed, were used in this
study to evaluate anti-oxidative effects of the beverage rather than puriﬁed extracts of
single compounds that have been used in various other in vitro studies. Pre-treatment of
Cac02 cells with the green tea decoction was effective in protecting against hydrogen
peroxide-induced DNA single strand. breaks as shown in by a signiﬁcant reduction in
percentage of comets. This result supports previous evidence demonstrating an anti-

oxidant effect of green tea (Yang, 2002).

In contrast to the results with the Comet assay, little effect was observed with

hydrogen peroxide treatment on the 8-OHdG content of Cac02 cells using the same dose

46

and duration of treatment. Concentration of 8-OHdG was similar to that of untreated
cells. This result might have been due to insufﬁcient dosage of the oxidizing agent.
Previous studies with hydrogen peroxide treatment to induce detectable DNA damage
typically range between 50 M to 2 mM in different cell types (Duthie and Dobson,
1999; Ivancsits et al., 2002). The study by Feng et al. (2002) involved measurement of 8-
OHdG by ELISA in RL-34 cells, a liver epithelial derived cell line. DNA damage was
induced by a glucose/ glucose oxidase (G/GO) system that continuously generated

hydrogen peroxide, the concentration of which was not reported.

The duration of hydrogen peroxide treatment used in this study was based on
viability of CacoZ cells at various time points. Viability remained unaltered even at 40
minutes, but a 20-minute incubation period was selected to be used in the Comet assay.
This treatment time was sufﬁcient to produce the desired effect, that is, production of
signiﬁcantly higher levels of single strand breaks compared to untreated cells. It is
possible that the 20 minute duration of hydrogen peroxide treatment of cells was enough
to cause signiﬁcantly higher levels of one type of DNA damage but not another namely,

8-OHdG.

HPLC coupled with electrochemical detection is the most commonly used
method to detect 8—OHdG in both human and in vitro studies, including those in cell
lines. There have been very few studies published to date that have employed ELISA to
measure this compound and fewer still are studies conducted in cell lines. There are no
previous reports that we know of that measured DNA damage in Cac02 cells speciﬁcally
and using the 8-OHdG kit that was used in this study. Perhaps this method was

unsuitablefor the model used in this experiment. This method is a monoclonal antibody

47

based assay. It can be speculated that the hydrogen peroxide impaired the detection
ability of the assay, perhaps by damaging the antibody binding sites and altering them in
such a way that they do not bind. The combination of conditions used in this study has
not been reported elsewhere and therefore results cannot be compared to any previously
published data.

Pre-treatment with green tea decoction had no effect on the 8-OHdG content of
CacoZ cells compared with cells treated with hydrogen peroxide alone. The 8-OHdG
content of cells pre-treated with black tea was higher than that in untreated cells as well
as cells treated with hydrogen peroxide alone, but the differences were not statistically
signiﬁcant. The differences in the concentration of tea as well as the experimental
models used in previous studies make it difﬁcult to compare those data with results from
this study. Tea decoction was obtained after a 5 minute steeping time, which is sufﬁcient
for extracting ahnost 90% of tea polyphenols into the water (Dashwood et al., 1999).
However, the undigested tea decoction was diluted to maintain uniformity with tea that
underwent the in vitro digestive process. The ﬁnal concentration of tea decoction that
cells were exposed to was 3.2% wt/vol. Previous studies have demonstrated anti-oxidant
effect of aqueous extracts of green tea. Administering a crude green tea extract prepared
by steeping 1:5 wt/vol of green tea leaves for 10 min decreased the expression of COX-2
enzyme and suppressed formation of neoplastic lesions in the colon of azoxymethane
treated male Wistar rats (Metz et al., 2000). J aved and Shukla (2000) used 2% aqueous
tea extract of green tea leaves to inhibit the onset of tumorigenesis when supplied as
drinking water in mice. There was also a reduction in the average number of tumors per
mouse. A 3% green tea aqueous extract serving as the sole source of drinking water for

10 days was protective against 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine

48

(PhIP) initiated carcinogenesis in Sprague Dawley rats (Lin et al., 1998). While these
concentrations are comparable to that which was used in this study, the cells were
subjected to a one time, short duration exposure to tea, which does not compare to the
long term feeding of tea in animal experiments. Therefore, it is probable that this dilution
of tea in our cellular model and the ﬁnal concentration of tea polyphenols, as well as
exposure time were responsible for the lack of antioxidant effect seen in this study. The
studies cited previously used carcinogens rather than oxidizing agents such as the
hydrogen peroxide, which was used in this study. The incubation time of 1h for pre-
treatment of cells also was perhaps not sufficient to alter effects observed with
subsequent treatment with the oxidizing agent.

To our knowledge there are no previous reports of studies that used tea decoction
to directly treat cells. There are some studies in which polyphenolic compounds present
in tea were applied directly to cells and an antioxidant effect demonstrated (Noroozi et
al., 1998; Erba et al., 1999; Aheme et al., 2000). However, tea used in this study,
prepared in the manner described and at that particular concentration, failed to show an
effect when compared to untreated cells and the effect of hydrogen peroxide. Our results
are in agreement with some studies that have been unable to show a protective effect of
polyphenols when tested in vitro (Sun et al., 2000). It is also to be noted that most
epidemiological studies in support of a beneﬁcial role of tea report a daily consumption
of tea that generally varies from 8-10 cups / day, and that a reduced risk for development
of chronic diseases was not observed when daily consumption was 1 cup / d. No
evidence was found for a protective effect even with regular consumption of tea (Nagano
et al., 2001). , In the present study, the same concentration of tea protected against single

strand breaks but not against 8-OHdG production.

49

Concentrations of 8-OHdG in cells pre-treated with green and black tea
decoctions that had been subjected to simulated gastric and intestinal digestion were
similar to those found with undigested tea. Although it is impossible to completely
mimic normal digestive conditions with an in vitro model, these results suggest that the
digestive enzymes as well as the pH changes involved in preparation of the digests had
no effect on the oxidative effect of tea. However, because hydrogen peroxide treatment
alone did not produce measurable DNA damage, a deﬁnitive conclusion with regard to
the effect of digestion is not possible.

In contrast to green tea, digested black tea produced a signiﬁcantly higher level of
8-OHdG in colon cells then did the experimental blank. The explanation for this result is
not readily apparent. No effect of digestion was observed on changes in 8-OHdG
production in~Cac02 cells when black tea decoction was subjected to the digestive
process. Black tea digest did not cause a signiﬁcant change in DNA damage when
compared to hydrogen peroxide treated cells. Results observed were similar to those with
undigested black tea decoction. Some interactions might have occurred between the
black tea and c0nditions of the simulated digestive process, which were different ﬁom
those with green tea. -

Black tea has been studied less extensively than green tea despite its being the
most abundantly consumed type of tea. However, there are data to show that black tea
theﬂavins are equally effective against oxidation (Leung et al., 2001). Some studies in
humans do not show a protective effect of black tea against cancer at low levels of
consumption. In fact, a study from Finland that assessed the relative risks of tea
consumption and colon cancer in middle aged men reported that subjects consuming 2 1

cup per day of black tea had an R of 2.09 compared to those that did not drink tea

50

suggesting a negative effect of black tea (Hartman et al., 1998). Studies that have shown
a positive association between tea drinking and risk for esophageal cancer have largely
been attributed to the high temperature of these beverages when consumed (Yang, 2002).

In contrast to result from human studies, Fadhel and Amran (2002) demonstrated
a signiﬁcant reduction in CC14 induced lipid peroxidation in liver, kidney and testes of
rats when a 0.7% boiling water infusion of black tea leaves was given as drinking water.
As was true for green tea, the extent of dilution of the black tea decoction to its ﬁnal
concentration (3.2% wt/vol) in the present study, and hence its polyphenolic content prior
to treatment of cells‘, could possibly have been the reason for no apparent effect on 8-
OHdG production.

Although green tea digested in the presence of cereal caused a signiﬁcantly
greater concentration of the oxidized base than in untreated cells and digest blank, it was
not higher than H202 treated cells or tea alone. Similarly, black tea digested with a
breakfast cereal, showed no signiﬁcant effect on 8-OHdG production. These results
suggest that cereal had no effect on the production of 8-OHdG in Cac02 cells. Literature
shows that iron promotes oxidation of DNA via the Fenton reaction (Toyokuni et al.,
1996). Iron reacts with H202 to yield more hydroxyl radicals, which are the most
damaging of the reactive oxygen species (Halliwell and Gutteridge, 1992). EGCG in
green tea has been suggested to be able to act in a pro-oxidant capacity, but only in
contact with unbound transition metal ions, such as iron and copper (Hu and Kitts, 2001).
However, there might not have been sufﬁcient amounts of iron in the cereal to exert such
an effect. In addition, the breakfast cereal contains other components, which could have

affected the anticipated effect of iron on the tea decoctions.~

5]

Compared to digests containing green tea alone, the presence of milk in the green
tea digest did not increase the 8-OHdG content of cells. However, the content of cells
pre-treated with this tea-milk digest was signiﬁcantly higher than that found in cells only
treated with H202, thus suggesting a possible pro-oxidant effect of this combination of tea
and milk. This aggravation of DNA damage in cells treated with milk-containing tea
digest was not observed in the case of black tea. Although it is well established that
protein from milk interacts with polyphenols (Loomis et al., 1966), it is to be noted that
the milk used in this experiment provided only a small amount of protein, since it was
based on the amount of milk that might be added to tea rather than to cereal. The
presence of protein is believed to result in binding of polyphenols leading to less effective
anti-oxidative action of tea. Thus it was postulated that milk would abolish an anti-
oxidative effect of tea by binding to polyphenols and thereby reduced DNA damage.
Since, an anti-oxidative effect was not demonstrated in this study, the increase in 8-
OHdG formation in cells treated with green tea-milk digest was unexpected and conﬂicts
with results of most studies. Previously published studies on the effect of addition of
milk to tea have been conducted mostly using black tea since in most cultures green tea is
not typically consumed with milk. Partially skimmed milk added to green tea during
digestion abolished the anti-mutagenic activity of green tea by more than 60 % when
evaluated by the Ames test (Krul et al., 2000). In this study, the addition of partially
skimmed milk to green tea signiﬁcantly increased 8-OHdG content of Cac02 cells.
Previous reports on the effect of milk on tea are conﬂicting. Arts et al., (2002)
demonstrated that a part of the total antioxidant capacity of green and black tea measured
with the Trolox equivalent antioxidant capacity (TEAC) assay, is ‘masked’ by interacting

with beta casein in milk. This ﬁnding suggests that food matrices inﬂuence the

52

effectiveness of tea as an antioxidant. In an in vitro experiment, addition of milk was
shown to have no effect on the relative antioxidant capacity of green and black tea and
other polyphenol containing beverages (Richelle et al., 2001). Additionally, the presence
of milk in black tea did not prevent the absorption of ﬂavonoids in humans (Hollman et
al., 2001). Interestingly, Langley-Evans (2000), documented that drinking black tea with
milk totally negates its in viva antioxidant potential as measured by FRAP assay in
healthy humans. In contrast, however, Leenen and colleagues (2000) reported that
addition of milk to tea does not abolish the signiﬁcant increase in plasma antioxidant
activity following ingestion of a single serving of black tea. Addition of milk to tea
reportedly had a greater protective effect compared to black tea alone by decreasing
mammary gland tumor multiplicity and volume in 7,12-Dimethylbenz [a] anthracene
(DMBA) and 2-amino-3-methyl-imidazo[4,5-f] quinoline (IQ) treated rats. Male Fisher
344 rats treated with azoxymethane in the same study showed decreased aberrant crypt
f0ci in the colon. These results suggest that milk can possibly potentiate the inhibitory
effects of black tea (Weisburger et al., 1997). Tewari et al. (2000) did not show an effect
for milk on the anti-oxidative property of tea. Milk was inhibitory on the anti-mutagenic
property of green and black tea in an in vitro digestive model, where partially skimmed
milk reduced the anti—mutagenic effect of black tea by 42 % (Krul et al., 2000).

These conﬂicting results demonstrate the difﬁculty in assigning a deﬁnitive role
for food matrices or dietary additives on the antioxidant characteristic of both green and
black tea. It is clear from the present study that green and black tea interacted differently
with additives, namely milk and cereal yielding different results for their impact on
oxidative damage in colon cells. Cac02 cells were treated with green tea that had been

digested in the presence of cereal had signiﬁcantly higher 8-OHdG production compared

53

to the effect of black tea. Similar results were obtained in the case of the addition of milk
to tea during the digestive process. The differential effects of the addition of milk to tea
during digestion could be attributed to the difference in the types of polyphenols present
in each. The major proportion of polyphenol in green tea is EGCG whereas theaﬂavins
are the most potent polyphenols in black tea Both have received recent recognition as
being protective against CVD and cancer (Yang, 1999). The galloyl moiety of both
theaﬂavins and catechins render them more potent as anti-oxidants compared to the non-
gallate forms (Leung etal., 2001). Although some studies have shown similar effects of
milk on both types of tea, others have not. Similarly, the difference in response observed
with addition of cereal could be attributed to this difference in cherrrical make-up
between green and black tea.

In conclusion, this study did not demonstrate a protective effect of green or black
tea. Because of the lack of increased 8-OHdG production in response to the H202
treatment used this experiment, the effect of tea is difﬁcult to interpret. However,
digestion appears to have no inﬂuence. Combination of cereal and tea did not modify 8-
OHdG formation, whereas the addition of rrrilk to green tea increased the 8-OHdG
content in cells suggestive of a pro-oxidant effect. However, this was not seen with black
tea. Signiﬁcant differences in results between green and black tea may be attributable to

the different types of ﬂavonoids present in the two teas.

54

SUMMARY AND CONCLUSIONS

Tea from the plant Camellia sinensis is a popular beverage that is consumed
worldwide and due to its polyphenolic content may potentially prevent development of
certain chronic diseases. A reduced risk of cancer at various sites has been associated
with consumption of tea in some populations. Studies in animals and cells lines have
demonstrated antioxidant activities of compounds extracted from green and black tea. A
drawback of these studies is the use single compounds and puriﬁed extracts and at levels
many fold higher than what would be encountered physiologically. Moreover, there are
very few data on the effect of other nutrients and dietary compounds on this beneﬁcial
property of tea.

This study was conducted to evaluate if tea prepared in the conventional method
of brewing would affect the potential cytoprotective characteristic of green and black tea.
It also was of interest to see if and how the digestive process, which involves changes in
pH and exposure to enzymes, might inﬂuence the antioxidant nature of tea.

The Caco2 cell line, a representation of the human intestine was selected to study
the effect of tea decoction or tea digested with or without the presence of rrrilk or cereal
on hydrogen peroxide induced oxidative DNA damage. A 4 g portion of green or black
tea leaves was steeped in boiling water for 5 min and the decoction was used to pre-treat
Cac02 cells prior to treatment with 500 uM H202. Tea decoction was subjected to in
vitro digestion and used similarly to pre-treat colon cells. The addition of cereal or milk
to tea during digestion was also included in the experiment. Cellular damage resulting
from hydrogen peroxide treatment was measured as DNA single strand breaks (Comet

assay) and as 8-hydroxy-2'-deoxyguanosine, a DNA base» adduct formed by the oxidation
of guanine. ‘

55

Hydrogen peroxide at 500 uM induced signiﬁcantly higher DNA damage in
Cac02 cells compared to untreated cells when tested by the Comet assay. Pre-treating
cells with green tea decoction prior to treatment with hydrogen peroxide caused a
signiﬁcant reduction in single strand breaks compared to the peroxide treated cells alone.
This experimental method was discontinued due to practical problems that limited its use
and interpretation of results.

In contrast to the effectiveness of the dose used in the comet assay, 500 uM H202
did not signiﬁcantly increase DNA damage in the form of 8-OHdG formation in Cac02
cells compared to untreated cells. The use of H202 at this concentration to induce DNA
damage in CacoZ cells to be measured as 8-OHdG formation by ELISA was not suitable
for the model used in this study.

Results for effect of pre-treatment with green tea decoction on DNA damage in
Cac02 cells was not signiﬁcantly different from the effect of hydrogen peroxide treatment
alone. The simulated digestive process did not alter the effect of tea decoction on 8-
OHdG formation in cells. The addition of a high iron breakfast cereal to tea during
digestion also had no inﬂuence on the effect observed with either H202 or with tea-
pretreatrnent alone. Addition of partially skimmed milk to the tea decoction during
digestion and pre-treatment of cells with this digest resulted in signiﬁcantly higher
concentration of 8-OHdG compared to hydrogen peroxide treated cells. This
concentration was not signiﬁcantly different from undigested tea. Similar effects in
Cac02 cells were observed with black tea treatments, but the addition of milk to black tea
decoction did not signiﬁcantly inﬂuence the formation of 8-OHdG.

Different effects were observed in green and black tea when cereal was added to

tea decoction during digestion. Treatment of Cac02 cells with green tea co-digested with

56

cereal prior to hydrogen peroxide treatment resulted in higher level of DNA damage
compared to black tea digested with cereal. Similar differences were seen between green
and black tea with the addition of milk.

Results of this study indicate that green tea decoction was protective against
hydrogen peroxide treatment in the Comet assay but neither black nor green tea
signiﬁcantly altered DNA base adduct formation compared to hydrogen peroxide
treatment in the 8-OHdG assay. However, the 8-OHdG content of cells treated with
500 uM hydrogen peroxide was similar to that in untreated cells making it difﬁcult to
make any deﬁnitive conclusions. Similar effects were obtained with the addition of
cereal or milk to both teas with the exception of green tea and milk, where an apparent
pro-oxidant effect was observed. Signiﬁcant differences were observed in 8-OHdG

formation between green and black tea with the addition of cereal or milk.

57

SUGGESTIONS FOR FUTURE RESEARCH

This study was an exploratory effort to elucidate the effect of digestion as well as
the presence of iron and protein during digestion on the antioxidant property of green and
black tea in Cac02 cells. One of the main limitations was the failure to produce DNA
damage with hydrogen peroxide in the 8-OHdG assay, which was necessary to correctly
interpret results. Therefore, the ﬁrst experiment that would need to be conducted to
clarify the ﬁndings of this study would be to test the dose response of H202 treatment to
induce 8-OHdG formation in Cac02 cells that would be signiﬁcantly higher compared to
no treatment (control). Once this has been established, the efﬁcacy of green and black
tea decoctions and tea digests in protecting against oxidative stress can be investigated.
In the event that the tea and tea digests still do not exhibit a protective effect against
oxidative stress with the strength used in the present study, it is conceivable that stronger
concentrations of tea might reduce the DNA damage caused by the hydrogen peroxide
treatment.

The addition of milk produced an apparent pro-oxidant effect when added to
green tea. If this result can be reproduced, it would be of interest to see the combined
effect of both milk and cereal in the tea digest, which might yield different results from
the addition of milk and cereal alone. The addition of puriﬁed sources of protein such as
casein and elemental sources of iron such as ferrous sulfate would be another approach to
clarify the results obtained, because the presence of other components in the milk and
cereal digests might have affected the outcomes in this study. The effect of addition of
other commonly consumed foods to tea can also be investigated. Of particular interest
would be those that are rich in copper, a transition metal that has been shown to induce

ﬂavonoids to behave as pro-oxidants in in vitro systems.

58

Experiments involving co-treatment of cells with tea decoctions and hydrogen
peroxide is likely to yield different results to that observed with pre-treatment. Although
Cac02 cells are colon cells, they exhibit characteristics of intestinal cells in culture and
have been used previously to test the efﬁcacy of antioxidant compounds. However, they
are derived from a colon adenocarcinoma and therefore already have substantial levels of
DNA damage (Kondo et al., 1999). The same experiment can therefore be conducted in
normal intestinal or colon cell lines to compare differences in effects on healthy and

cancerous cells.

59

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