Phase IIa chemoprevention trial of green tea polyphenols in high-risk individuals of liver cancer: Modulation of urinary excretion of green tea polyphenols and 8-hydroxydeoxyguanosine

Article (PDF Available)inCarcinogenesis 27(2):262-8 · February 2006with35 Reads
DOI: 10.1093/carcin/bgi147 · Source: PubMed
Abstract
Modulation of urinary excretion of green tea polyphenols (GTPs) and oxidative DNA damage biomarker, 8-hydroxydeoxyguanosine (8-OHdG), were assessed in urine samples collected from a randomized, double-blinded and placebo-controlled phase IIa chemoprevention trial with GTP in 124 individuals. These individuals were sero-positive for both HBsAg and aflatoxin-albumin adducts, and took GTP capsules daily at doses of 500 mg, 1000 mg or a placebo for 3 months. Twenty-four hour urine samples were collected before the intervention and at the first and third month of the study. Urinary excretion of 8-OHdG and GTP components was measured by HPLC-CoulArray electrochemical detection. The baseline levels of 8-OHdG and GTP components among the three groups showed homogeneity (P > 0.70), and a non-significant fluctuation was observed in the placebo group over the 3 months (P > 0.30). In GTP-treated groups, epigallocatechin (EGC) and epicatechin (EC) levels displayed significant and dose-dependent increases in both the 500 mg group and 1000 mg group (P < 0.05). The 8-OHdG levels did not differ between the three groups at the 1 month collection, with medians of 1.83, 2.08 and 1.86 ng/mg-creatinine for placebo, 500 and 1000 mg group, respectively (P = 0.999). At the end of the 3 months' intervention, 8-OHdG levels decreased significantly in both GTP-treated groups, with medians of 2.02, 1.03 and 1.15 ng/mg-creatinine for placebo, 500 mg and 1000 mg group, respectively (P = 0.007). These results suggest that urinary excretions of EGC and EC can serve as practical biomarkers for green tea consumption in human populations. The results also suggest that chemoprevention with GTP is effective in diminishing oxidative DNA damage.
Phase IIa chemoprevention trial of green tea polyphenols in high-risk
individuals of liver cancer: modulation of urinary excretion of green
tea polyphenols and 8-hydroxydeoxyguanosine
Haitao Luo
1
, Lili Tang
1
, Meng Tang
1
, Madhavi Billam
1
,
Tianren Huang
1
,
2
, Jiahua Yu
2
, Zhongliang Wei
3
,
Yongqiang Liang
3
, Kaibo Wang
3
, Zhen-Quan Zhang
2
,
Lisheng Zhang
2
and Jia-Sheng Wang
1,
1
The Institute of Environmental and Human Health and Department of
Environmental Toxicology, Texas Tech University, PO Box 41163, Lubbock,
TX 79409-1163, USA,
2
Guangxi Cancer Institute, Nanning, Guangxi, China
and
3
Fusui Liver Cancer Institute, Fusui, Guangxi, China
To whom correspondence should be addressed. Tel: þ1 806 8850320;
Fax: þ1 806 8852132;
Email: js.wang@ttu.edu
Modulation of urinary excretion of green tea poly-
phenols (GTPs) and oxidative DNA damage biomarker,
8-hydroxydeoxyguanosine (8-OHdG), were assessed in
urine samples collected from a random ized, double-
blinded and placebo-controlled phase IIa chemoprevention
trial with GTP in 124 individuals. These individuals were
sero-positive for both HBsAg and aflatoxin–albumin
adducts, and took GTP capsules daily at doses of 500 mg,
1000 mg or a placebo for 3 months. Twenty-four hour urine
samples were collected before the intervention and at
the first and third month of the study. Urinary excretion
of 8-OHdG and GTP components was measured by
HPLC-CoulArray electrochemical detection. The base-
line levels of 8-OHdG and GTP components among the
three groups showed homogeneity (P 4 0.70), and a non-
significant fluctuation was observed in the placebo group
over the 3 months (P 4 0.30). In GTP-treated groups,
epigallocatechin (EGC) and epicatechin (EC) levels dis-
played significant and dose-dependent increases in both
the 500 mg group and 1000 mg group (P 5 0.05). The
8-OHdG levels did not differ between the three groups
at the 1 month collection, with medians of 1.83, 2.08 and
1.86 ng/mg-creatinine for placebo, 500 and 1000 mg group,
respectively (P ¼ 0.999). At the end of the 3 months’ inter-
vention, 8-OHdG levels decreased significantly in both
GTP-treated groups, with medians of 2.02, 1.03 and
1.15 ng/mg-creatinine for placebo, 500 mg and 1000 mg
group, respectively (P ¼ 0.007). These results sugges t
that urinary excretions of EGC and EC can serve as prac-
tical biomarkers for green tea consumption in human
populations. The results also suggest that chemopreven-
tion with GTP is effective in diminishing oxidative DNA
damage.
Introduction
Primary liver cancer, mainly hepatocellular carcinoma (HCC),
is one of the most common cancers in Asia, Africa, and in
populations of Asian- and Hispanic-Americans (1,2). Because
of its poor prognosis, HCC has a mortality approaching its
morbidity, and is the third cause of cancer deaths in the world
(3,4). Eighty percent of the world’s HCC cases arise in the
developing world in Southeast Asia and sub-Saharan Africa
(5), where the major etiologic factors have been identified as
hepatitis B virus (HBV) infection and dietary aflatoxin (AF)
exposure (6,7), with synergistic effects suggested by several
epidemiological studies (8,9). Primary prevention strat egies,
such as HBV vaccination in infants and AF control in food, can
diminish the exposure to major risk factor s and have offered
the best hope for reducing HCC morbidity in these com mu-
nities; however, outcomes may require decades to appear.
Currently, the great challenge in HCC prevention and control
is how to reduce the risk in individuals who have already
been exposed to both etiological risk factors for decades.
Chemoprevention has been proposed as a promising strategy
to help these high-risk individuals.
Green tea polyphenols (GTPs) have been shown safe and
effective as chemopreventive agents in various in vitro bio-
assays and in vivo animal models for inhibition of carcinogen-
induced mutagenesis and tumorigenesis at several target organ
sites, including AFB
1
-induced liver tumors (10–12). GTP is the
secondary metabolite in tea plants, and accounts for 30–36%
weight of the water extractable materials in tea leaves. The
major GTP components include ()-epigallocatechin gallate
(EGCG), ()-epigallocatechin (EGC), ()-epicatechin gallate
(ECG), and ()-epicatechin (EC), with EGCG being the most
abundant (11). Though the safety and efficacy of GTP are
consistent in most animal studies, human epidemiological
studies have so far generated controversial results (13). Some
studies found no association or even worse, a positive associ-
ation between tea drinking and cancer risk, while others
revealed a reduced risk of cancer in the esophagus, stomach,
lung and liver with green tea consumption (14–18).
Several mechanisms have been proposed for the anti-
carcinogenic effect of GTP, with the well-accepted one that
GTP can capture and detoxify reactive oxygen species (ROS)
produced in the process of carcinogen metabolism, inflam-
mation, aerobic respiration and exposure to background radi-
ations (19). ROS attack all macromolecules including lipids,
proteins and DNA. The addition of hydroxyl radical (OH) to
the C8-position of guanine produces C8–OH adduct radical
(20), which is subsequently converted to 8-OH-guanine
(8-OH-Gua) by a one-electron oxidation (21). While damaged
lipids and proteins can be removed by metabolic turnover
of molecules, impaired DNA has to be repaired in situ,or
destroyed by apoptotic processes, if not to result in mutations.
In humans, the 8-OH-Gua glycosylase (OGG1) is the pri-
mary enzyme for the repair of 8-OH-guanine adduct in a
Abbreviations: AF, aflatoxin; EC, epicatechin; EGC, epigallocatechin; GTP,
green tea polyphenols; HCC, hepatocellular carcinoma; HBV, hepatitis B
virus; 8-OHdG, 8-hydroxydeoxyguanosine; ROS, reactive oxygen species.
Carcinogenesis vol.27 no.2
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Oxford University Press 2005; all rights reserved. 262
Carcinogenesis vol.27 no.2 pp.262–268, 2006
doi:10.1093/carcin/bgi147
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short-patch base-excision repair (BER) (22). The excised form
of 8-OH-Gua is 8-hydroxy-2
0
-deoxyguanosine (8-OHdG),
which is excreted into urine without further metabolism and
is stable for a significant time (23,24). Thus, urinary 8-OHdG
generally reflects the whole body’s oxidative DNA damage
and repair, and becomes a putative biomarker for oxidative
stress (24). Detection of urinary 8-OHdG provides a sensitive
and non-invasive means to evaluate the efficacy of chemo-
prevention.
Lack of objective data on the amount of tea consumption has
hindered the precise evaluation of the association betwee n tea
ingestion and human cancer risk in questionnaire-based epi-
demiological studies (25–28). It has been proposed that the
quantitative measurement of GTP components in human body
fluids is a more appro priate way to reflect green tea consump-
tion in prospective epidemiological studies (18,29). However,
the best GTP component to serve as an exposure biomarker
has not yet been determined and/or validated at the population
level, and a precise evaluation of the role of GTP in cancer risk
will most likely come from a prospective human intervention
study.
Our work presented here is based on a randomized, double-
blinded, and placebo-controlled phase IIa chemopreventio n
trial with GTP in a HCC high-risk population (30). The urine
samples collected from this trial were analyzed for GTP com-
ponents to validate biomarkers for tea consumption, and urin-
ary 8-OHdG levels were assessed as a surrogate endpoint to
evaluate the efficacy of GTP intervention in these high-risk
individuals.
Materials and methods
Materials
GTP was obtained from the US–China joint venture Shili Natural Product
Company, (Guilin, Guangxi) with purity 498.5%, and encapsulated by the
Guangxi Pharmaceutical Company (Nanning, Guangxi). Authentic stand-
ard GTP components and 8-OHdG, and b-glucuronidase, sulfatase, ascorbic
acid and creatinine detection kit were purchased from Sigma–Aldrich
Chemical Company (St Louis, MO). Oasis
HLB cartridges were products
of Waters Corporation (Milford, MA). All organic solvents used were of
high-performance liquid chromatography (HPLC) grade. Other chemicals
and reagents were purchased at the commercially highest degree of purity
available.
Study design and procedure
The design, clinical outcome and baseline biomarker data of this phase IIa GTP
chemoprevention trial has been previously described (30). The overall study
design is shown in Figure 1. Briefly, 1200 blood samples were screened and
124 voluntary residents were enrolled into this trial. The recruiting criteria
include healthy adults with positive serum HBsAg and detectable AF–albumin
adducts by radioimmunoassay, among others (30). Informed consent was
obtained from each participant before they were randomly assigned to three
study groups, and baseline blood and urine samples were collected before the
intervention began. Randomization was successful as no significant differ-
ences with regard to age, gender and baseline AF–albumin levels were found
between groups (30). Participants were instructed to take four capsules daily
containing either GTP 500 mg (low-dose, n ¼ 42), GTP 1000 mg (high-dose,
n ¼ 41) or starch 1000 mg as placebo (control, n ¼ 41). The doses of 500 and
1000 mg GTP were chosen to be equivalent to two and four cups of tea drink,
respectively. Follow-up visits were taken every other day at the participant’s
house to record possible adverse-effect complaints and to count the remaining
capsules for adherence assessment. No severe adverse-effects were recorded
according to clinical tests of blood and urine samples at each collection,
including blood counts, blood chemistry, alanine aminotransferase (ALT),
aspartate aminotransferase (AST), urinary protein, glucose, blood and others
(30). An excellent person–time compliance (99.5%) was achieved, and no
other consumption of tea or tea products was reported for any participant in
this trial (30).
In addition to regular epidemiological questionnaires, blood samples (5 ml
for serum and 5 ml for plasma) and 24 h urine samples were collected at
1 month and 3 months of the intervention. Serum, plasma and blood cells were
immediately separated and stored at 20
C in the village clinics. Twenty-four
hour urine samples were collected in the morning, noon and evening in 1 day,
and kept in amber bottles containing ascorbic acid (20 mg/ml) and EDTA
(0.1 M). Aliquots of urine samples (50 ml) were treated with 500 mg ascorbic
acid and 12.5 mg EDTA for analysis of GTP components and 8-OHdG
analysis. All samples were shipped frozen to Texas Tech University and the
laboratory personnel who performed analysis were blinded to sample sources.
This study was approved by Institutional Review Boards of Texas Tech
University and Guangxi Cancer Institute for human subject protection. Sample
collection, storage and shipment complied with guidelines of both Chinese and
US governments.
Analysis of urinary GTP components
The protocol for urinary GTP analysis was modified from methods previously
described by Yang et al. (29,31). Briefly, thawed urine samples were
centrifuged and 1 ml supernatant taken for a 1 h digestion with 500 U of
b-glucuronidase and 2 U of sulfatase (Sigma) to release conjugated tea
polyphenols. The urine samples were then extracted twice with ethyl acetate.
Organic phases were pooled, dried in vacuo with a Labconco Centrivap
concentrator (Kansas City, MO), reconstituted in 15% acetonitrile, and ana-
lyzed with the ESA HPLC-CoulArray system (Chelmsford, MA). The system
consists of double Solvent Delivery Modules (Model 582 pump), Autosampler
(Model 542) with 4
C cool sample tray and column oven, CoulArray Electro-
chemical Detector (Model 5600A), and an Operating Computer. The HPLC
column was an Agilent Zorbax reverse-phase column, Eclipse XDB-C
18
(5 mm,
4.6 250 mm). The mobile phase included buffer A (30 mM NaH
2
PO
4
/ACN/
THF ¼ 98/1.8/0.2, pH 3.35) and buffer B (15 mM NaH
2
PO
4
/ACN/THF ¼ 30/
63/7, pH 3.45). Flow rate was set at 1 ml/min and the gradient started from 4%
buffer B, to 24% B at 24 min, to 95% B at 35 min, kept at 95% until 42 min,
dropped to 4% at 50 min, and maintained at 4% until 59 min. Authentic
standards were prepared with ascorbic acid and aliquots of the mixture stock
were stored at 80
C for 1 month’s use. Calibration curves for individual GTP
component were generated separately, and EGC, EC, EGCG and ECG were
eluted at around 14, 21, 24 and 29 min, respectively. The electrochemical
detector was set at 90, 10, 70 and 150 mV potentials, with the main peaks
appearing at 10 mV (EGC), 70 mV (EC, EGCG) and 150 mV (ECG). Quality
assurance and quality control procedures were taken during analyses, including
analysis of authentic standards for every set of five samples and simultaneous
analysis of spiked urine sample daily. The limits of detection were 1.0 ng/ml
urine for EC and EGC and 1.5 ng/ml urine for EGCG and ECG, respectively.
Urinary GTP components were adjusted by creatinine level to eliminate the
variation in urine volume.
Analysis of urinary 8-OHdG
Protocol for urinary 8-OHdG analysis was modified from the method
described by Renner et al. (32). Briefly, 8-OHdG was extracted from 1 ml
urine with the Oasis
HLB 3 cc (60 mg) cartridge (Waters) following the
manufacturer’s instructions. The eluents were dried under ultra-pure N
2
stream
and reconstituted in buffer (10 mM ammonium acetate in 2% MeOH, pH 4.3)
for analysis with the HPLC-ECD system, which was the same as previously
described in the analysis of urinary GTP. The HPLC column for 8-OHdG
analysis was Waters YMC basic column (S3 mm, 4.6 150 mm). The
mobile phase consists of buffer A (10 mM ammonium acetate, pH 4.3)
Fig. 1. Overall study design of the phase IIa chemoprevention trial.
Modulation of urinary excretion of GTP and 8-OHdG
263
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and buffer B (methanol). Flow rate was kept at 0.8 ml/min and a linear gradient
(0–40% MeOH in 15 min) was applied for chromatographic separation with
the peak of 8-OHdG eluted at 9.5 min. The CoulArray Detector was set at
270, 300, 330 and 360 mV, with the highest peak appearing at the 330 mV
channel. Authentic standard 8-OHdG (Sigma–Aldrich) was used for qualifica-
tion by retention times and response patterns, and quantification by calibration
curves. Similar quality assurance and quality control procedure were applied as
described in analysis of urinary GTP, and the limit of detection for 8-OHdG
was 1 ng/ml urine. The amount of 8-OHdG was also adjusted by urinary
creatinine level for analysis and report.
Analysis of urinary creatinine
Urinary creatinine level was determined colorimetrically with a Diagnostics
Creatinine Kit (Sigma–Aldrich) following the manufacturer’s instructions.
Absorbance at 500 nm was recorded by a DU640 VIS/UV spectrophotometer
(Beckman Coulter).
Statistical analysis
Due to the nature of repeated measurements within each participant in this
study, the longitudinal data analysis was applied with a multi-level model for
change. As only three waves of data were available, a linear change over time
was assumed in the following model (33):
Level-1 model: Y
ij
¼p
0i
þp
1i
TIME
ij
þe
ij
e
ij
Nð0, s
2
e
Þ
Level-2 model: p
0i
¼g
00
þg
01
GROUP
i
þj
0i
p
1i
¼g
10
þg
11
GROUP
i
þj
1i
j
0i
j
1i

N
0
0

,
s
2
0
s
01
s
10
s
2
1

,
where the level-1 model represents the ‘within-person change’ in biomarker
(urinary GTP or 8-OHdG) levels over TIME, and the level-2 model represents
the ‘between-person differences in change’ in biomarker levels and associates
the participant’s trajectory (intercept and slope) with the predictor, GROUP.
The fixed effects, g
00
, g
01
, g
10
, g
11
, capture systematic differences according
to values of GROUP and are of particular interest here. The model was fitted
with MIXED program in SPSS 11.0 (SPSS Chicago, IL) implementing
maximum likelihood method for parameters’ estimation [(34), http://www.
ats.ucla.edu/stat/examples/alda/]. To monitor closely when the efficacy
of intervention appears, non-parametric ANOVA (Kruskal–Wallis test)
was also applied at all three collections with SPSS to give a cross-sectional
perspective of this study. A two-tailed P-value 50.05 is reported as
significant.
Results
Modulation of urinary GTP biomarkers
Major GTP components in chromatographs were identified
and integrated at corresponding maximum-respons e channels
of the CoulArray detector. EGC and EC were readily detected
in most samples. The recovery rate with spiked authentic
standards at different levels was averaged 75.2 3.9% for
urinary EGC and 95.1 1.6% for urinary EC. The coefficient
of variance was 513% for EGC and 11% for EC.
Changes in urinary excretion of EGC and EC levels over the
baseline, 1 month or 3 month samples after intervention are
shown in Figures 2 and 3. The homogeneity of urinary excre-
tion of EGC and EC levels at baseline is demonstrated by
cross-sectional ANOVA (P 4 0.80). Analysis of these urinary
GTP data with the multi-level model for change is shown in
Table I. The initial level in placebo group (GROUP ¼ 0) was
308.8 ng/mg creatinine for EGC (g
00
¼ 308.8, P ¼ 0.000) and
55.3 ng/mg creatinine for EC (g
00
¼ 55.3, P ¼ 0.337). For
GTP-treated groups (GROUP ¼ 500 or 1000 mg), the initial
levels of both EGC ( g
01
un-modeled) and EC (g
01
¼0.02,
P ¼ 0.745) were not significantly different to placebo group.
The slopes for both EGC (g
10
¼ 20.10, P ¼ 0.478) and EC
(g
10
¼1.08, P ¼ 0.976) in placebo group (see Table I) were
not significantly different from 0, which means generally no
change in urinary GTP levels in this group were detected
over the 3 month period. For GTP-treated groups, however,
the slopes were significant. For urinary EGC (g
11
¼ 0.09,
P ¼ 0.014), the slope was 0.09 ng/mg creatinine/month higher
Fig. 2. Urinary excretion of epigallocatechin (EGC). EGC was analyzed for
three groups at baseline, 1 month and 3 months. Levels were adjusted by
urinary creatinine levels. Upper panel: Baseline EGC levels in three groups
show homogeneity (P ¼ 0.832). Middle panel: 1 month EGC levels show
nearly significant elevation in both GTP-treated groups (P ¼ 0.113). Lower
panel: 3 month EGC levels remained non-significant but higher in both
GTP-treated groups (P ¼ 0.119).
Fig. 3. Urinary excretion of epicatechin (EC). EC was analyzed for three
groups at baseline, 1 month and 3 months. Levels were adjusted by urinary
creatinine levels. Upper panel: Baseline EC levels in three groups show
homogeneity (P ¼ 0.919). Middle panel: 1 month EC levels show significant
elevation in both GTP-treated groups (P ¼ 0.004). Lower panel: 3 month EC
levels remained significantly higher in both GTP-treated groups (P ¼ 0.008).
H.Luo et al.
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for every milligram GTP intervention. In two treated groups
(GROUP ¼ 500 or 1000 mg), the slope increased 45 ng/mg
creatinine/month for the 500 mg group and 90 ng/mg
creatinine/month for the 1000 mg group, as compared to the
zero slope in the placebo group. For urinary EC (g
11
¼ 0.11,
P ¼ 0.065), there is a marginally significant 0.11 ng/mg
creatinine/month increase in the slope for every milligram
GTP intervention. So the slope of EC increas ed 55 ng/mg
creatinine/month for the 500 mg group and 110 ng/mg
creatinine/month for the 1000 mg group, as compared to the
zero slope in the placebo group. These results suggest that,
while urinary GTP levels remained unchanged in the placebo
group over the 3 month period, the GTP- treated groups showed
significant, dose-dependent increases in urinary EGC and EC
levels over time. The prototypical trajectories in the three
groups for both EGC and EC levels were shown in Figure 4.
The increased levels of urinary EGC and EC were also found
significant at both 1 month and 3 month collection, as shown in
Figures 2 and 3.
Modulation of urinary 8-OHdG levels
Several commercially available cartridges, including the
LiChrolut EN cartridge, the Waters Sep-Pak cartridge and
the Waters Oasis column, were tested for concentration and
purification of urinary 8-OHdG. The Waters Sep-Pa k (C18
cartridge) can barely retain spiked 8-OHdG in urine. The
LiChrolut EN cartridge had a reco very 50% for spiked
8-OHdG. The notes in the Waters Oasis
HLB (polymer-
based column) had a better recovery (470%) and was tested
for its capacity to bind 8-OHdG with a range of 1–6000 ng
spiked urine samples and was selected to concentrate and
purify 8-OHdG for the analysis of all urine samples. The
limit of detection for the Oasis
HLB (60 mg column) was
1 p.p.b. Upon CoulArr ay detector, 8-OHdG has a maximum
response at 330 mV channel, which was chosen to quantify this
biomarker in urine samples. Both retention time and response
patterns were scrutinized for identifying target peaks, and
almost all samples have well-separated and detectable
8-OHdG peaks. The coefficient of variance for the 8-OHdG
analyses was 515%.
Changes of urinary excretion of 8-OHdG levels over the
baseline, 1 month or 3 month samples after intervention are
Fig. 4. Prototypical trajectories for urinary GTP levels in three groups.
Urinary levels of EGC and EC were fitted to a multi-level model for change
with maximum likelihood estimations. Both model fits found no significant
change in placebo group over time (P 4 0.470), but significant and
dose-dependent elevation in EGC (P ¼ 0.014) and EC (P ¼ 0.065) in
GTP-treated groups. Upper panel: model estimation of urinary EGC levels in
three groups over time with respective prototypical trajectories. Lower panel:
model estimation of urinary EC levels in three groups over time with
respective trajectories.
Fig. 5. Urinary excretion of 8-OHdG. 8-OHdG was analyzed for three
groups at baseline, 1 month and 3 months. Levels were adjusted by urinary
creatinine levels. Upper panel: Baseline 8-OHdG levels in three groups show
homogeneity (P ¼ 0.742). Middle panel: 1 month 8-OHdG levels remained
similar in three groups (P ¼ 0.999). Lower panel: 3 month 8-OHdG levels
were significantly diminished by GTP intervention (P ¼ 0.007).
Table I. Parameter estimation in multi-level model for biomarker levels
Parameter EGC EC 8-OHdG
Intercept, p
0i
Intercept g
00
308.8 ( P ¼ 0.000) 55.3 (P ¼ 0.337) 3.7 (P ¼ 0.002)
GROUP g
01
0.02 (P ¼ 0.745) 0.002 (P ¼ 0.277)
Slope, p
1i
Intercept g
10
20.1 (P ¼ 0.478) 1.08 (P ¼ 0.976) 0.524 (P ¼ 0.384)
GROUP g
11
0.09 (P ¼ 0.014) 0.11 (P ¼ 0.065) 0.002 (P ¼ 0.036)
Modulation of urinary excretion of GTP and 8-OHdG
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shown in Figure 5. The homogeneity of urinary 8-OHdG levels
at baseline was again proved by cross-sectional ANOVA (P 4
0.74). Analysis of urinary 8-OHdG data with the multi-level
model for change is also shown in Table I. The initial 8-OHdG
level in placebo group (GROUP ¼ 0) was 3.7 ng/mg creatinine
(g
00
¼ 3.7, P ¼ 0.002). For GTP-treated groups (GROUP ¼
500 or 1000), the initial levels (g
01
¼ 0.002, P ¼ 0.277) were
not significantly different from the placebo group.
The slope for 8-OHdG (g
10
¼ 0.524, P ¼ 0.384) in the
placebo group was not significantly different from 0, suggest-
ing an unchanged 8-OHdG level in this group over the 3 month
period. However, for GTP-treat ed groups ( g
11
¼0.002,
P ¼ 0.036), the slope significantly decreased 0.002 ng/mg
creatinine/month for every milligram GTP intervention. For
treated groups (GROUP ¼ 500 or 1000), the slope decreased
1.0 ng/mg creatinine/month for the 500 mg group and
2.0 ng/mg creatinine/month for the 1000 mg group, as com-
pared to that in the placebo group. The prototypical trajectories
in three groups for 8-OHdG are shown in Figure 6. Though
the baseline levels of 8-OHd G seem a little different, they were
homogeneous as proved by model fittings (P ¼ 0.277) and
the slope of placebo group was not statistically different from a
zero slope (P ¼ 0.384, Table I). These results suggest that,
while urinary 8-OHdG levels remained unchanged in the pla-
cebo group over the 3 month period, the GTP-treated groups
had a significant, dose-dependent decrease in 8-OHdG levels
over time. The diminished 8-OHdG levels were also found
significant at 3 month collection by cross-sectional ANOVA
(P ¼ 0.007), as shown in Figure 5.
Discussion
Cancer chemoprevention is defined as the use of drugs, diet
or dietary supplements at the earlier stages of carcinogenesis
to prevent initiation of cancer, or to retard or delay the pro-
gression of cancer (35). In high-risk individuals, initiation is
usually assumed, and the goal of chemoprevention is to retard
or delay promotion and/or progression. Currently, HCC is the
third leading cause of cancer mortali ty in the world (3–5), and
the major etiological risk factors were identified as chronic
infection with HBV and dietary exposure to AF (36). While
the precise mechanism of HCC formation is poorly under-
stood, one common pathway is the generation of oxidative
stress, especially ROS (37,38). To target ROS in HCC
high-risk individuals, GTP, a naturally occurring antioxidant,
is mechanistically appropriate with well-known safety,
efficacy and popularity.
Humans have consumed tea for 45000 years, and currently
it is still the most commonly consumed beverage worldwide
(11,39). GTP has been shown to inhibit carcinogenesis in the
skin, lung, esophagus, stomach, liver, small intestine, pan-
creas, colon and mammary gland in various animal models,
demonstrating its inhibitory activity toward multiple stages of
carcinogenesis (14,18). In humans, however, inconsistent res-
ults were reported with respect to the role of GTP in cancer risk
(13). Lack of objective measures is a common flaw in ques-
tionnaire-based investigations where the level of tea ingestion
was subjectively classified according to individuals’ memor-
ies. Urinary biomarkers for tea consumption have practical
significances in epidemiological studies and the measurement
of various urinary GTP components has been established (25).
Validation of these components as biomarkers in high-risk
individuals with a controlled GTP intervention, as described
in this study, is a high priority in this research field.
In this study, only EGC and EC were readily detected in
most urine samples, and were dose-dependent with the dose
protocol of intervention, which was consistent with previous
reports (25,31,40). Though no tea drinking was reported in the
study population, a background level of urinary EGC and EC
was detected, which may suggest GTP sources other than tea
or tea products in this area, such as vegetables and fruits.
Nevertheless, GTP-treated groups, as compared to the placebo
group, showed significant and dose-dependent increases in
both EGC and EC levels, which supported urinary excre-
tion of EGC and EC as reliable biomar kers for green tea
consumptions.
It has been demonstrated that GTP could prevent oxygen free
radical-induced hepatocyte lethality and inhibit carcinogen-
induced liver oxidative DNA damage (41,42). Humans
are ordinarily being attacked by ROS, and DNA constantly
damaged (43). The oxidative adduct form of guanine,
8-OH-Gua, is not merely a consequence of oxidative damage,
but also a risk factor for further genetic mutations if kept
in situ. The physicochemical property of 8-OH-Gua affects
transcription and replication, and facilitates mispairing with
dA and dT (mostly causing G!T substitution) (44,45). 8-OH-
Gua also produces base substitution errors at adjacent
upstream and downstream template sites (46). Even in the
absence of mutations, epigenetic effects have been noticed
to affect certain gene expressions: the presence of 8-OH-Gua
in promoter elements can affect transcription factor binding, as
a single 8-OH-Gua moiety in the promoter region of AP-1
completely prevented transcription factor binding and further
gene expression (47).
8-OH-Gua is mainly repaired by base-excisions and the
excised product, 8-OHdG, is exclusively excreted into urine
(23). Because of its stability in urine (48), its non-invasive
sampling, its absence of artifacts as encountered in DNA
extraction (24), and more importantly its etiological role in
mutations and gene expressions, urinary 8-OH-dG has been
proposed to be an appropriate, intermed iate biomarker of
both oxidative DNA damage and disease outcome (22). In this
study, all participants have been exposed to AFB
1
and the
community has been chronically infected with HBV for dec-
ades, both of which have been reported to increase the forma-
tion of ROS in their pathogenic pathways (37,38), thus the
oxidative burden in these high-risk individuals is presumably
overwhelming the body’s defense/repair ability, and an
accumulation of massive oxidative DNA damage is expected.
This was confirmed by the baseline data with significant
Fig. 6. Prototypical trajectories for urinary 8-OHdG levels in three groups.
Urinary levels of 8-OHdG were fitted to a multi-level model for change with
maximum likelihood estimations. Fitted model found no significant change
8-OHdG levels in placebo group over time (P 4 0.380), but significant and
dose-dependent level changes (P ¼ 0.036) in GTP-treated groups.
H.Luo et al.
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8-OHdG levels in all three groups, ranging from 3.7 to
5.7 ng/mg creatinine. This baseline level was comparable to
previously reported 8-OHdG levels in other high-risk popula-
tions, such as heavy smokers (24,49). Smokers had a higher
level of urinary 8-OHdG, 1.95 mmol/mol creatinine, which
corresponds to a level of 4.88 ng/mg creatinine (24). The
urinary 8-OHdG level ranged from 5 to 20 ng/mg creatinine
for eight male smokers in another report (49). The accumu-
lated oxidative DNA damage seems to be abundant and con-
sistent in study participants, as demonstrated by no significant
decreases in 8-OHdG levels after 1 month intervention with
GTP. This is different from the previous report that green tea
significantly reduced the oxidative burden and decreased urin-
ary 8-OHdG levels in both smokers and non-smokers after
7 days’ treatment (50). It seems that combinative oxidative
damage caused by HBV and AF is more extensive and more
difficult to be modulated. Fortunately , intervention with GTP
for 3 months’ significantly reduced 8-OHdG levels in these
high-risk individuals, which confirmed the efficacy of GTP in
chemoprevention of combinative oxidative damage. Previous
studies found that GTP modulates body’s antioxidant–oxidant
balance through changing enzyme profiles, in addition to its
free radical-scavenging and metal chelating abilities,
e.g. inhibition of oxidative stress-increasing enzymes such
as inducible nitric oxide synthase, lipoxygenases, cyclooxy-
genases and xanthine oxidase, or induction of antioxidant
enzymes like glutathione S-transferase, glutathione peroxi-
dase, catalase and superoxide dismutase (51). A relatively
longer period may be necessary for the induction/inhibition
of these enzymes. Significant reduction of 8-OHdG levels
after 3 months’ intervention, as shown in this study, favors
the importance of modulation of enzyme profiles. This will be
further examined in the phase III long-term study that is
on-going in this high-risk population.
In summary, results of this study suggest that chemo-
prevention with GTP can effectively reduce 8-OHd G levels,
the oxidative DNA damage biomarker. Urina ry excretion
of EGC and EC was the validated biomarker for green tea
consumption.
Acknowledgements
This study was supported by research grants ES11442 from the NIEHS and
CA90997 from the NCI.
Conflict of Interest Statement: None declared.
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    • "The experimental data on GTP provided the impetus to translate this strategy to human clinical trials. In an initial study in an aflatoxinexposed high-risk group in Guangxi, People's Republic of China, the effects of GTP was assessed by analysis of blood and urine samples collected from a randomized, doubleblinded , placebo-controlled Phase IIa chemoprevention trial [144]. Blood serum of all participants contained aflatoxin– albumin adducts at the outset. "
    [Show abstract] [Hide abstract] ABSTRACT: Collectively, liver cancer, including hepatocellular carcinoma (HCC) and cholangiocarcinoma, accounts for 9.1 % of all reported cancer deaths and is the second most common cause of cancer mortality worldwide Ferlay et al. (Int J Cancer 136:E359–86, 2012). The incidence of liver cancer varies enormously globally and unfortunately the burden of this nearly always fatal disease is much greater in the less economically developed countries of Asia and sub-Saharan Africa World Cancer Report 2014 (International Agency for Research on Cancer, 2014).
    Chapter · Jan 2016 · International Journal of Molecular Sciences
    • "Garlic possesses some anti-inflammatory and anti-arthritic properties which may play a role in the treatment of inflammatory and arthritic diseases [64]. Green tea, when supplemented, demonstrated protective effects along with diminished oxidative DNA damage in a randomized, double-blinded and placebo-controlled phase trial [65]. Oolong tea extract (8 g/d) supplementation for 6 weeks in obese /overweight women reduced body fat content and body weight and improved lipid metabolism [66]. "
    Full-text · Dataset · Apr 2015 · International Journal of Molecular Sciences
    • "Recent literature indicates that green tea extract, including GTCs, may inhibit certain types of microsomal cytochrome P450 (CYP), and may not lead to drug-induced liver injury when a drug and green tea are administered simultaneously [78]. A randomized, double-blind, placebo-controlled phase IIa chemoprevention trial demonstrated that GTCs have antioxidant effects in individuals who have several risk factors for HCC, and this may suggest that chemoprevention with GTCs is an effective strategy for diminishing oxidative DNA damage [79]. Conversely, Jin et al. [80] reported that GTCs did not significantly reduce HCC incidence or HCC-related mortality in a review of four clinical studies. "
    [Show abstract] [Hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC), which is a common malignancy worldwide, usually develops in a cirrhotic liver due to hepatitis virus infection. Metabolic syndrome, which is frequently complicated by obesity and diabetes mellitus, is also a critical risk factor for liver carcinogenesis. Green tea catechins (GTCs) may possess potent anticancer and chemopreventive properties for a number of different malignancies, including liver cancer. Antioxidant and anti-inflammatory activities are key mechanisms through which GTCs prevent the development of neoplasms, and they also exert cancer chemopreventive effects by modulating several signaling transduction and metabolic pathways. Furthermore, GTCs are considered to be useful for the prevention of obesity- and metabolic syndrome-related carcinogenesis by improving metabolic disorders. Several interventional trials in humans have shown that GTCs may ameliorate metabolic abnormalities and prevent the development of precancerous lesions. The purpose of this article is to review the key mechanisms by which GTCs exert chemopreventive effects in liver carcinogenesis, focusing especially on their ability to inhibit receptor tyrosine kinases and improve metabolic abnormalities. We also review the evidence for GTCs acting to prevent metabolic syndrome-associated liver carcinogenesis.
    Full-text · Article · Mar 2015
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