Effect of different doses of un-fractionated green and black tea extracts on thyroid physiology

Abstract

Tea is a rich source of polyphenolic flavonoids including catechins, which are thought to contribute to the health benefits of it. Flavonoids have been reported to have antithyroid and goitrogenic effect. The purpose of this study was to evaluate whether high doses of green and black tea have a harmful effect on thyroid physiology. Un-fractionated green and black tea extracts were administered orally to male rats for 30 days at doses of 1.25 g%, 2.5 g% and 5.0 g%. The results showed that green tea extract at 2.5 g% and 5.0 g% doses and black tea extract only at 5.0 g% dose have the potential to alter the thyroid gland physiology and architecture, that is, enlargement of thyroid gland as well as hypertrophy and/or hyperplasia of the thyroid follicles and inhibition of the activity of thyroid peroxidase and 5(')-deiodinase I with elevated thyroidal Na+, K+-ATPase activity along with significant decrease in serum T3 and T4, and a parallel increase in serum thyroid stimulating hormone (TSH). This study concludes that goitrogenic/antithyroidal potential of un-fractionated green tea extract is much more than black tea extract because of the differences in catechin contents in the tea extracts.

Full text

Effect of different doses of
un-fractionated green and black tea
extracts on thyroid physiology
Amar K Chandra, Neela De and
Shyamosree Roy Choudhury
Abstract
Tea is a rich source of polyphenolic flavonoids including catechins, which are thought to contribute to the
health benefits of it. Flavonoids have been reported to have antithyroid and goitrogenic effect. The purpose
of this study was to evaluate whether high doses of green and black tea have a harmful effect on thyroid phy-
siology. Un-fractionated green and black tea extracts were administered orally to male rats for 30 days at doses
of 1.25 g%, 2.5 g% and 5.0 g%. The results showed that green tea extract at 2.5 g% and 5.0 g% doses and black
tea extract only at 5.0 g% dose have the potential to alter the thyroid gland physiology and architecture, that is,
enlargement of thyroid gland as well as hypertrophy and/or hyperplasia of the thyroid follicles and inhibition of
the activity of thyroid peroxidase and 50-deiodinase I with elevated thyroidal Naþ,Kþ-ATPase activity along
with significant decrease in serum T3 and T4, and a parallel increase in serum thyroid stimulating hormone
(TSH). This study concludes that goitrogenic/antithyroidal potential of un-fractionated green tea extract is
much more than black tea extract because of the differences in catechin contents in the tea extracts.
Keywords
catechins, green tea, black tea, thyroid, thyroid peroxidase, Naþ,Kþ-ATPase, 50-deiodinase I
Introduction
Tea is a pleasant, popular, socially accepted and eco-
nomical drink that is enjoyed everyday by hundreds of
millions of people across all continents.
1
The pleasing
astringent taste and refreshing boost it provides is so
deep-pervasive that its potential health benefits and
medicinal properties are often overlooked. Ongoing
scientific exploration points that certain potential
health benefits derived from tea have important impli-
cations on human heath.
2
Although there are thousands of tea varieties, tea is
generally divided into three groups based on the pro-
cess of fermentation they undergo during processing.
These are unfermented tea (white and green tea),
semi-fermented tea (oolong tea) and fully fermented
tea (black tea). Green tea and black tea are both
derived from the tea leaves of Camellia sinensis.To
produce green tea, freshly harvested leaves are
steamed to prevent fermentation, yielding a dry, sta-
ble product. They are rich in flavonoids and have the
highest quantity of tea catechins that are chemically
defined as flavan-3-ols. To produce black tea, the
fresh leaves are allowed to wither, decreasing their
moisture content, until their weight is *55%of the
original leaf weight. The withered leaves are then
rolled and crushed, initiating fermentation of poly-
phenols. This fermentation converts catechin to thea-
flavins and thearubigins that give the characteristic
aroma and color of the black tea and consequently less
catechin content.
3
In general, green tea contains about
30%w/w of catechins in the dry leaves. The major
catechins, which are found in abundant proportion,
are ()Epigallo catechin gallate (EGCG), ()Epi-
gallo catechin (EGC), ()Epicatechin (EC) and ()
Endocrinology and Reproductive Physiology Laboratory,
Department of Physiology, University of Calcutta, Kolkata,
West Bengal, India
Corresponding author:
Amar K Chandra, Endocrinology and Reproductive Physiology
Laboratory, Department of Physiology, University of Calcutta,
University College of Science and Technology, 92, Acharya
Prafulla Chandra Road, Kolkata 700 009, West Bengal, India
Email: amark_chandra@yahoo.co.in
Human and Experimental Toxicology
30(8) 884–896
ªThe Author(s) 2010
Reprints and permission:
sagepub.co.uk/journalsPermissions.nav
DOI: 10.1177/0960327110382563
het.sagepub.com

Epicatechin gallate (ECG) with ()Catechin gallate
(CG). Other compounds obtainable in tea are the
flavonols (quercetin, kaempferol, myricitin and rutin),
caffeine, phenolic acids, theanine and flavour
compounds.
4
By oral intake, EGCG, the pure com-
pound, has an LD
50
of more than 1 g/kg body weight
(1390 mg/kg) in rat. Black tea contains less tea
catechins (3%10%w/w), while theaflavins and
thearubigins account for about 2%6%w/w and
10%20%w/w of the dry weight of the leaves,
respectively.
5
Green tea is commonly consumed in
China, Japan and Eastern Asia, while black tea is
mainly brewed in European countries and India. The
intake of catechins can be expected to be higher in the
Asiatic countries and the health effects of green tea
may be more apparent when examined in the Asian
communities.
6
Tea is a source of a wide range of phytochemicals
that are digested, absorbed and metabolized by the
body and that tea constituents exert their effects at the
cellular level.
1
Abundant experimental and epidemio-
logic evidences provides a convincing argument that
polyphenolic antioxidants present in green and black
tea can reduce several chronic diseases, especially
cardiovascular disease and cancer.
7
Despite its enormous usefulness and potential health
benefits, flavonoids emerge as phytochemicals of
great concern due to their antithyroidal and goitrogenic
effect. It has previously been reported that the con-
sumption of flavonoids and some phenolic acids by
experimental animals induced enlargement and
histological changes in the thyroid gland.
8-12
Previous
study reported that after a 13-weeks oral administration
of polyphenone-60 (P-60), which is green tea extract
(GTE) catechins, it was found that this treatment at
high dose induced goiters in rats.
13,14
However,
studies on the un-fractionated tea extracts on thyroid
physiology are not available though tea is used as
whole beverage rather than taking any single compo-
nent of it, so it is very essential to study the effect of
un-fractionated green and black tea extracts on rats.
In this present investigation, the effects of green and
black tea consumption at high doses on the thyroid
physiology will be explored to find out whether there
is goitrogenic effect of tea extracts on normal rats. For
this purpose, dose- or concentration-dependent effects
of un-fractionated green and BTEs without any specific
purification of possible candidate compounds on thyr-
oid peroxidase (TPO), 50-deiodinase I (50-DI) and
sodium-potassium adenosine triphosphatase (Naþ,
Kþ-ATPase) activities, thyroid gland architecture and
serum levels of thyroid hormones were examined in
vivo.
Materials and methods
Materials
DTE, Tris-HCl and Na
2
ATP were purchased from
Sisco Research Laboratories (SRL), Mumbai, India.
Ouabain, PTU, T4, bovine serum albumin, EDTA,
MnCl
2
were purchased from Sigma Chemical Company,
Steinheim, Germany. SDS was purchased from LOBA
Chemie Pvt. Ltd, Mumbai, India. Goat anti-rabbit
g-globulin, polyethylene glycol and chloramine T were
purchased from Sigma, St Louis, MO, USA.
Green tea was collected from Institute of
Himalayan Bioresource Technology (IHBT), Palam-
pur, Himachal Pradesh, India. The black tea was
collected from Tea Research Association, Tocklai
Experimental Station, Jorhat, Assam, India. The com-
ponents of tea were analyzed by the method of HPLC
gradient elution (Hitachi HPLC, Darmstad, Ger-
many). The composition of green tea denotes contents
of EC 1.55%, EGCG 9.00%, ECG 4.8%, EGC
5.0%, Caffeine 2.38%as mentioned by the pro-
ducer. The composition of the black tea as provided
by the producer was theaflavins (TF) 1.56%, thear-
ubigins (TR) 11.95%and total catechins (TC) 
4.80%. Preparation of aqueous extract of green tea
was done following the method of Wei et al.
15
Briefly,
2.5 g green tea leaf was added to 100 mL of boiling
water and was steeped for 15 min. The infusion was
cooled to room temperature and then filtered. The tea
leaves were extracted a second time with 100 mL of
boiling water and filtered and two filtrates were
combined to obtain a 1.25-g%tea aqueous extract
(1.25 g%tea leaf/100 mL water). By the same proce-
dure 2.5 g%and 5.0 g%green tea aqueous extract was
prepared. The BTEs were also prepared by the same
procedure. These solutions of both tea extracts were
prepared freshly everyday.
Animals and treatment
In the present study, 3-months-old adult male albino
rats of Sprague Dawley strain weighing 200 +10 g
were used that were obtained from Indian Institute
of Chemical Biology (IICB), Kolkata. The animals
were maintained as per national guidelines and proto-
cols, approved by the Institutional Animal Ethics
Committee (PHY/CU/IAEC/07 dated 25.07.2007).
The animals were housed in clean polypropylene
Chandra AK et al. 885

cages and maintained in a controlled environment at
temperature 22C+2C and relative humidity
(40%60%) in an animal house with constant
12 hours light and 12 hours dark schedule. The ani-
mals were fed on standardized diet which consisted
of 70%wheat, 20%Bengal gram, 5%fish meal
powder, 4%dry yeast powder, 0.75%refined til oil
and 0.25%shark liver oil and water ad libitum.
16
In the 30 days treatment, the experimental animals
were divided into seven groups of eight animals each.
The first group was administered sterile distilled
water orally by gavage as vehicle and considered as
control. Next three groups of animals received un-
fractionated GTE orally at doses of 1.25 g%(mild
dose), 2.5 g%(moderate dose) and 5.0 g%(high
dose), respectively, daily. Another three groups
received the same doses of un-fractionated black tea.
The GTE and BTE were administered to animals
orally by gavage once daily at a dose of 1 mL/100 g
body weight. All the animals were sacrificed 24 hours
after the last treatment (i.e. during 9 am to 10 am on
the day of experiment to avoid any discrepancy that
may arise for diurnal variation) following protocols
and ethical procedures. Blood samples were collected
and serum separated for hormone assay.
Body weight and food consumption
The body weights (g) of the experimental animals
were recorded on the first day before the start of treat-
ment (initial) and the day of sacrifice (final). Food
consumption of the animals per cage was recorded
daily during the period of treatment.
Thyroid weight
Just after sacrifice, the thyroid glands were taken out,
trimmed off the attached tissues and weighed. The
relative weight of thyroid gland (mg) was expressed
per 100 g body weight.
Histological study
Immediately after removal, the thyroid gland of each
rat was fixed in 10%neutral buffered formalin,
embedded in paraffin and sections were stained with
hematoxylin & eosin (HE) staining and examined
under a light microscope.
Thyroid peroxidase assay
Thyroid peroxidase activity was measured by the
method of Alexander.
17
For TPO activity, 10%
homogenate was prepared using thyroid tissues
collected from the sacrificed animals, in phosphate
buffer (pH 7.2, 100 mM) and sucrose solution
(500 mM) at 4C. Homogenisation was carried out in
a glass homogeniser (Potter-Elvehjem) for 4560 sec
at 4400 gand about 15 strokes/min. The homogenate
was centrifuged at 1000 gfor 10 min. This low-
speed supernatant was further centrifuged at 10,000 g
for 10 min at 4C to get the mitochondrial fraction.
The microsomal fraction containing most of peroxi-
dase activity was obtained by centrifuging the
post-mitochondrial supernatant at 1,05,000 gfor 1 hour.
Immediately after centrifugation, the precipitate
was solubilized in phosphate buffer (pH 7.2).
Thyroid peroxidase activity was measured in a 1 mL
cuvette containing 0.9 mL acetate buffer (pH 5.2,
50 mM), 10 mL potassium iodide (1.7 mM), 20 mL
microsomal fraction of thyroid tissue, and freshly
prepared 20 mL hydrogen peroxide (0.3 mM) was
added lastly to start the reaction for assaying the TPO
activity (DOD/min/mg protein) in spectrophotometer
(UV-1240 Shimadzu) at 353 nm. The pooled sample
was assayed in duplicate.
Thyroidal Naþ,Kþ-ATPase assay
Thyroidal Naþ,Kþ-ATPase activity was measured
by a modification of the method of Esmann et al.
18
In brief, microsomal fraction of thyroid tissue homo-
genate was incubated in reaction mixtures of
(i) 30 mM imidazole HCl, 130 mM NaCl, 20 mM
KCl, 4 mM MgCl
2
and (ii) 30 mM imidazole HCl,
4 mM MgCl
2
and 1 mM ouabain (Sigma Chemical
Co., St. Louis, MO 63178, USA) at pH 7.4 for 60 min
at 0C. The reaction was started by addition of 4 mM
Tris-ATP at 37C and stopped with 0.1 mL of 20%
SDS after 10 min. The inorganic phosphate (Pi) liber-
ated was determined by reading the absorbance at 850
nm in a UV-mini1240, Shimadzu, Japan, by the
method of Baginski et al.
19
The enzyme activity was
expressed as nmols of Pi liberated per hour per mg
protein calculated from a standard curve of potassium
dihydrogen phosphate. The pooled sample was
assayed in duplicate.
50-deiodinase I (5’-DI) 50-DI assay
Iodothyronine 50-deiodinase type I (5’-DI) 50-DI
activity was measured according to the method of
Ko¨dding et al.,
20
with slight modifications. Briefly,
a substrate solution of 0.1 M Tris-HCl buffer (pH
7.4), 3 mM EDTA and 150 mM DTE containing 0.4
886 Human and Experimental Toxicology 30(8)

mM T4 and 100150 mg thyroid tissue protein in a
final volume of 400 mL was incubated at 37C for
30 min. The monodeiodination reaction of T4 to T3
was terminated by addition of 800 mL ice-cold abso-
lute ethanol, followed by shaking for 8 min at 4C.
The reactants were then centrifuged at 10,500 g at
4C for 8 min and the ethanol supernatants were
collected for measurement of T3 content. For all
samples, values for zero time were prepared by
adding the thyroid tissue to the substrate containing
T4 after the addition of alcohol. The concentration
of T3 in the ethanolic extract after 0 and 30 min of
incubation were estimated by enzyme-linked
immunosorbent assay (ELISA). The activity of
50-DI was calculated as the difference of the 0 and
30 min values and expressed in terms of pmoles T3
formed/mg protein. The pooled sample was assayed
in duplicate.
The validity of the assay method has been justi-
fied by preincubation of the sample with the 50-DI
inhibitor, propylthiouracil (PTU), that resulted in
>50%inhibition of the enzymatic activity. It needs
tobementionedherethatconversionofT4torT3
by 50-DI cannot proceed under such simulated con-
ditions, as rT3 formation can occur only under high
pH and substrate concentration, unlike T4 to T3
monodeiodination, as found in our experimental
condition.
Protein estimation
Proteins were estimated by the method of Lowry
et al.,
21
using bovine serum albumin (BSA) as the
standard protein.
ELISA of serum T3 and T4
Just before sacrifice, blood samples were collected for
each rat under ether anesthesia and the serum was
separated for the assay of T3 and T4. All the samples
for measurement were preserved at 20C. Total
serum triiodothyronine and thyroxin were assayed
using ELISA kits obtained from RFCL Limited, India
(Code no HETT 1108 and HETF 0908 respectively).
The sensitivity of the T3 and T4 assays were
0.04 ng/mL and 0.4 mg/dL, respectively.
Radioimmunoassay (RIA) of thyroid
stimulating hormone
Serum levels of thyroid stimulating hormone (TSH)
were assayed by radioimmunoassay (RIA) using
reagents supplied by Rat Pituitary Distribution and
National Institute of Diabetes and Digestive and Kid-
ney Diseases (NIDDK) (California, USA). NIDDK-
rTSH was iodinated using the chloramine-T method
22
with carrier free
125
I. Serum rat TSH antiserum
(NIDDK–anti-rTSH–RIA-6) had no significant cross
reactivity with rat Growth Hormone (rGH), rat
Follicle stimulating hormone (rFSH), rat Luteinizing
hormone (rLH) and rat Prolactin (rPRL). The assay
was calibrated with rat TSH reference preparation
(NIDDK-rTSH-RP3).
125
I was purchased from the
Bhabha Atomic Research Center (BARC), Mumbai,
India. Goat anti-rabbit g-globulin (Cambridge
Nuclear, Sigma) and polyethylene glycol (Sigma)
were used as the second antibody to precipitate the
antibody-bound TSH. Radioactivity was counted on
the Gamma counter (Electronic Corporation of India
Ltd) and the results were expressed as ng/mL. The
sensitivity of the TSH assay was 0.01 ng/mL, and the
intra- and interassays coefficients of variation were
8.2%and 6%, respectively.
Statistical analysis
Results were expressed as mean +standard devia-
tion. One-way analysis of variance (ANOVA) test
was first carried out to test for any differences
between the mean values of all groups. If differ-
ences between groups were established, the values
of the treated groups were compared with those
of the control group by a comparison ttest. A
value of p< 0.05 was interpreted as statistically
significant.
23
Results
Body weight and food consumption
During the course of the present study, it was
observed that the body weight of the control animals
increased progressively throughout the period of
investigations and showed a net body weight gain
of þ31.8%. However, the net body weight gain of
the animals treated at high dose with GTE was notice-
ably less and was þ26.7%as compared to black
teatreated group of animals þ28.1%(Table 1).
During the course of GTE and BTE treatment, food
consumption of the experimental animals was
recorded (Table 2). Food consumption was almost
same in all the groups (mild, moderate and high-
dose groups) of experimental animals in comparison
to control animals.
Chandra AK et al. 887

Thyroid weight
Relative weight of thyroid glands were significantly
increased after green tea administration in a dose-
dependent manner as compared to the control group
of animals, whereas, the relative weight of thyroid
glands increased only after high dose of black tea
exposure for 30 days (Figure 1).
Histological changes
Histological assessments performed on thyroid section
of various groups were presented in Figures 2 and 3.
Thyroid appeared normal both grossly and microsco-
pically in both mild dose (1.25 g%) of green and black
teatreated groups (Marked B). However, green tea
treatment at moderate dose (2.5 g%) daily for 30 days
caused diffuse hypertrophy and/or hyperplasia of thyr-
oid follicular epithelial cells, with less amount of col-
loidal material and irregular shaped follicles having
decreased luminal spaces with tall cuboidal follicular
epithelium when compared with those of control and
the same dose (2.5g %) of black tea (Marked C). Green
tea treatment at high dose (5.0 g%) daily produced
large colloid-filled follicles with tall columnar epithe-
lium that occupied most of the thyroid tissue. In addi-
tion, the presence of desquamated epithelial cells and
mononuclear cell infiltration inside the follicles were
observed (Marked D). Thyroid sections of control
(Marked A) and the mild and moderate BTE-treated
Weight of thyroid gland
0
2
4
6
8
10
12
14
mg/100g body weight
Gr 5.0%Control
Bl 1.25%
Gr 1.25%
Bl 2.5%
Gr 2.5%
Bl 5.0%
a
a
a
c
d
Figure 1. Changes in the relative thyroid gland weight of
rats subjected to green and black tea extracts at different
doses. Each bar denotes mean +SD, n¼8. One-way
analysis of variance (ANOVA) test followed by a multiple
comparison ttest was performed. Significance at p< 0.05.
a
Control versus other groups;
c
green tea 2.5 g% versus
black tea 2.5 g%;
d
green tea 5.0 g% versus black tea 5.0 g%.
Gr ¼green tea extract treatment and Bl ¼black tea
extract treatment.
Table 1. Change in the body weight (g) of experimental animals subjected to different doses of green and black tea
extracts treatment
a
Group
Green tea extract treatment Black tea extract treatment
Initial weight Final weight Initial weight Final weight
Control 201 +7.4 265 +6.2 201 +7.4 264 +2.5
(% gain in body weight) (þ31.8%) (þ31.4%)
1.25 g% 204 +6.3 268 +5.6 202.1 +6.5 272.1 +6.0
b
(% gain in body weight) (þ31.4%) (þ34.7%)
2.5 g% 204.1 +6.5 263.5 +8.5 201.3 +2.4 262.5 +1.3
(% gain in body weight) (þ29.0%) (þ30.4%)
5.0 g% 202.5 +7.6 256.6 +3.8
b
203.5 +4.5 260.6 +3.4
b
(% gain in body weight) (þ26.7%) (þ28.1%)
a
Data are presented as the mean +SD, n ¼8. One-way analysis of variance (ANOVA) test followed by a multiple comparison ttest
was performed.
b
Significantly different by ANOVA at p< 0.05 when compared to control.
Table 2. Effects of green and black tea extract on food
consumption of rats for 30 days treatment
a
Group
Green tea extract
treatment
Black tea extract
treatment
g/animal/day g/animal/day
Control 24 +1.2 25 +1.6
1.25 g% 25 +1.1 26 +1.9
2.5 g% 26 +1.3 25 +2.4
5.0 g% 24 +2.1 27 +1.5
a
Data are presented as the mean +SD.
888 Human and Experimental Toxicology 30(8)

groups (Marked B and C respectively) showed no his-
topathologic features. However, BTE treatment only at
high dose (5.0 g%) causes mild hypertrophy and/or
hyperplasia of thyroid follicular epithelial cells lined
by columnar epithelium.
Thyroid peroxidase assay
Figure 4 compares the effect of green and black tea
administration at different doses on thyroid peroxi-
dase activity in adult albino male rats. Thyroid perox-
idase activity was decreased significantly in a dose-
dependent manner after green tea administration. No
significant changes in the activity of TPO were
observed at either mild or moderate doses (1.25 g%
and 2.5 g%) of black tea treatment when compared
with the control group, while treatment with high
doses (5.0 g%) of black tea for 30 days caused a more
prominent decrease in the enzyme activity.
Thyroidal Naþ,Kþ-ATPase assay
Figure 5 compares the level of thyroidal Naþ,
Kþ-ATPase in animals to different treatments. The
Figure 2. Photomicrographs of paraffin-embedded hematoxylin and eosin (H&E)-stained rat thyroid sections. A, Rat
thyroid section (400) from control animals. B, Thyroid section (400) from green tea-treated animals (Dose I).
C, Thyroid section (400) from green tea-treated animals (Dose II). D, Thyroid section (400) from green tea-
administered animals (Dose III). C ¼colloid, F ¼follicle of thyroid.
Chandra AK et al. 889

thyroidal Naþ,Kþ-ATPase activity was signifi-
cantly increased after administration of green tea
at moderate and high doses (2.5 g%and 5.0 g%
daily) for 30 days when compared to control. But
after the black tea treatment only at high dose
(5.0%) thyroidal Naþ,Kþ-ATPase activity was
increased significantly, whereas, at moderate dose
(2.5%), no significant changes were found. How-
ever, no change in the activity of Naþ,Kþ-ATPase
was observed in either 1.25 g%green or black tea–
treated groups.
Thyroidal 50-deiodinase I assay
Figure 6 provides data on thyroidal 50-deiodinase I
(50-DI) activity in control, green and black tea adminis-
tered at different doses in experimental animals. Green
tea treatment caused a statistically significant inhibition
in the activity of 50-DI in a dose-dependent manner,
while in black tea, inhibition in the 50-DI enzyme activ-
ity was found at a high dose(5.0 g%). However, no such
significant changes were observed in the animals treated
with black tea at mild and moderate doses.
Figure 3. Photomicrographs of paraffin-embedded hematoxylin and eosin (H&E)-stained rat thyroid sections. A, Rat
thyroid section (400) from control animals. B, Thyroid section (400) from black tea-treated animals (Dose I).
C, Thyroid section (400) from black tea-treated animals (Dose II). D, Thyroid section (400) from black
tea-administered animals (Dose III). C ¼colloid, F ¼follicle of thyroid.
890 Human and Experimental Toxicology 30(8)

Thyroid hormone level
Figures 7 and 8 show serum T3 and T4 level in control
and green and black tea administered animals at dif-
ferent doses. The serum T3 and T4 levels were
significantly decreased in a dose-dependent manner
in green tea administered animals as compared to con-
trol rats. However, serum T3 level was decreased sig-
nificantly only at high dose (5.0 g%) of black tea
treatment as compared to control.
Serum TSH level
Figure 9 shows that serum TSH level was signifi-
cantly increased in the green teatreated groups at
moderate and high doses (2.5 g%and 5.0 g%) over the
control values. However, no such significant changes
were observed in the animals treated with green tea at
the mild dose (1.25 g%) and black tea at the different
doses (1.25 g%, 2.5 g%and 5.0 g%) for 30 days.
Discussion
The present study demonstrated that un-fractionated
GTE exposure at medium (2.5 g%) and high (5.0
g%) dose as used in this investigation reduced the net
gain in body weight in adult albino rats when compared
with control group, even though the average daily food
intake did not differ between the groups during study
period. However, un-fractionated BTE exposure at
5.0 g%dose for 30 days as used in this investigation
reduced the net gain in body weight in treated rats
when compared with control group, but at dose of
1.25 g%and 2.5 g%weight gain was not significantly
affected. These findings are in line with previous work,
where it was shown that green tea has a potential role in
body weight control; in addition, caffeine and theanine
have been found to strengthen polyphenol effects on
body weight control and fat accumulation in mice.
24
Recently, it has been reported that oral administration
of GTE caused significant decrements in both the visc-
eral and hepatic fat accumulation of non-obese rats fed
Thyroid peroxidase activity
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
ΔOD/min/mg protein
Control Gr 1.25% Gr 2.5% Gr 5.0%
Bl 1.25% Bl 2.5% Bl 5.0%
a
a
a
c
d
b
Figure 4. Effect of green and black tea extracts at
different doses on thyroid peroxidase (TPO) activity in
rats. Each bar denotes mean +SD of three pooled
samples. Each pool containing a mixture of three thyroid
glands isolated from three individual rats. The assay was
repeated twice. One way analysis of variance (ANOVA)
test followed by a multiple comparison ttest was
performed. Significance at p< 0.05.
a
Control versus
other groups;
b
green tea 1.25 g% versus black tea 1.25 g%;
c
green tea 2.5 g% versus black tea 2.5 g%;
d
green tea
5.0 g% versus black tea 5.0 g%. Gr ¼green tea extract
treatment and Bl¼black tea extract treatment.
Thyroidal Na+,K+-ATPase activity
0
0.5
1
1.5
2
2.5
3
nmol Pi/min/mg protein
Control Gr 1.25% Gr 2.5% Gr 5.0%
Bl 1.25% Bl 2.5% Bl 5.0%
a
a
a
c
d
Figure 5. Effect of green and black tea extracts at
different doses on thyroidal Naþ,Kþ-ATPase activity in
rats. Each bar denotes mean +SD of three pooled
samples. Each pool containing a mixture of three thyroid
glands isolated from three individual rats. The assay was
repeated twice. One-way analysis of variance (ANOVA)
test followed by a multiple comparison ttest was
performed. Significance at p< 0.05.
a
Control versus
other groups;
c
green tea 2.5 g% versus black tea 2.5 g%;
d
green tea 5.0 g% versus black tea 5.0 g%. Gr ¼green tea
extract treatment and Bl ¼black tea extract treatment.
Chandra AK et al. 891

a normal diet and the tea catechin treatment caused a
decrease in the serum levels of cholesterol and bile
acids, showing that the tea catechins have a notable
effect on lipid metabolism in non-obese subject as well
as obese ones.
25
It has been further shown that lipid-
lowering effect of black tea in hyperlipidaemic rats is
through reactivation of lipoprotein lipases, increased
faecal excretion of cholesterol and bile acids.
26
The synthesis of thyroid hormones is the major
function of thyroid gland. The main regulatory
enzyme for thyroid hormones biosynthesis is thyroid
peroxidase which catalyzes the binding of iodine to
tyrosyl residues on thyroglobulin and the subsequent
coupling of iodotyrosyl residues required for the for-
mation of iodothyronine residues. The inhibition of
TPO-catalyzed reactions results in decrease in serum
levels of thyroid hormones, which leads to compensa-
tory increased secretion of TSH by the anterior pitui-
tary. The increased levels of TSH provide a growth
stimulus to the thyroid and it has been proposed that
a prolonged stimulus can select for clones of follicular
cells with the potential for transformation.
27
Green tea
exposure decreased the activity of thyroid peroxidase
in a dose-dependent manner, whereas, black tea expo-
sure decreased the same only at a high dose (5.0 g%).
No significant changes were found in the activity of
TPO after administration of black tea at mild and
moderate doses (1.25 g%and 2.5 g%), respectively.
Available literature shows that dietary flavonoids
have an antithyroidal effect and also to be goitrogenic.
Although flavonoids are reported to exert antithyroid
effects through a variety of mechanisms, reports on
the antithyroidal effects of catechins are limited.
Black tea contains high concentrations of theaflavins
and thearubigins and relatively low concentrations of
catechins, which are well-known natural flavonoids.
Divi and Doerge,
28
reported that catechins inhibited
TPO (IC
50
¼36.4 +3.86 mM). A probable mechanism
of action may relate to the ability of phenolic com-
pounds with a free resorcinol (metahydrophenol) moi-
ety to inhibit TPO. The proposed mechanism of action
for enzyme inhibition as found in the present study
involves the conversion of thyroid peroxidase to a free
radical that reacts with resorcinol moiety and produces
a flavonoid radical.
29
The flavonoid radical could
covalently bind to the catalytic amino acid residues
5'-deiodinase I activity
0
2
4
6
8
10
12
pmol T3/mg protein
Control Gr 1.25% Gr 2.5% Gr 5.0%
Bl 1.25% Bl 2.5% Bl 5.0%
a
a
a
c
d
Figure 6. Effect of green and black tea extracts at
different doses on thyroid 50-deiodinase I (50-DI) activity in
rats. Each bar denotes mean +SD of three pooled
samples. Each pool containing a mixture of three thyroid
glands isolated from three individual rats. The assay was
repeated twice. One-way analysis of variance (ANOVA)
test followed by a multiple comparison ttest was
performed. Significance at p< 0.05.
a
Control versus
other groups;
c
green tea 2.5 g% versus black tea 2.5 g%;
d
green tea 5.0 g% versus black tea 5.0 g%. Gr ¼green tea
extract treatment and Bl ¼black tea extract treatment.
Serum T3 level
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
ng/ml
Control Gr 1.25% Gr 2.5% Gr 5.0%
Bl 1.25% Bl 2.5% Bl 5.0%
a
a
a
c
d
Figure 7. Effect of green and black tea extracts at
different doses serum T3 level in rats. Each bar denotes
mean +SD, n¼8. One-way analysis of variance
(ANOVA) test followed by a multiple comparison ttest
was performed. Significance at p< 0.05.
a
Control versus
other groups;
c
green tea 2.5 g% versus black tea 2.5 g%;
d
green tea 5.0 g% versus black tea 5.0 g%. Gr ¼green tea
extract treatment and Bl ¼black tea extract treatment.
892 Human and Experimental Toxicology 30(8)

on the enzyme, leading to enzyme inactivation.
Through their inhibitory activity in thyroid
peroxidase, they can cause elevated TSH levels, which
promote thyroid gland growth and thyroid
dysfunction.
The most striking finding in this study was that
un-fractionated GTE increased the activity of Naþ,
Kþ-ATPase in a dose-dependent manner. Naþ,
Kþ-ATPase activity was also increased at a dose of
5.0 g%BTE. The first step in the synthesis of thyroid
hormones is the uptake of iodide from the extracellu-
lar fluid by thyroid follicular cells. The iodide is con-
centrated in the follicular cells by an active transport
mechanism, the so-called ‘Sodium-iodide symporter’
(NIS), which is energy consuming, connected to a
Naþ,Kþ-ATPase activity and stimulated by TSH.
NIS-mediated iodide transport is therefore inhibited
by the Naþ,Kþ-ATPase inhibitor ouabain as well
as by the competitive inhibitors thiocyanate and per-
chlorate.
30
Naþ,Kþ-ATPase is composed of two
subunits in equimolar ratios. These are the asubunit
with a molecular mass of *113 KDa and the
smaller glycosylated bsubunit with a protein portion
accounting for 35 KDa of the overall molecular mass
of 55 KDa. Isoforms exist for both a(a1, a2 and a3)
and b(b1, b2 and b3) subunits. Previous studies
strongly suggest that hypothyroidism increases the
number of Naþ,Kþ-ATPase subunits (a1 and b1)
in the rat thyroid gland.
31
In the present study,
increased Naþ,Kþ-ATPase activity can be attributed
due to development of a morphological as well as
functional hypothyroidism that develop under the
influence of catechins present in the un-fractionated
GTE and BTE.
Most of the circulating T3 originates from the
extra-thyroidal tissues. The peripheral deiodination
of T4 to T3, taking place mainly in the liver, kidney
and thyroid, is dependent on 50-monodeiodinase I
activity. Un-fractionated green tea as well as black tea
(only at high dose) exposure reduced significantly the
enzymatic activity of 50-monodeiodinase I, which
suggested that tea catechin decreased the rate of
conversion of T4 into T3. Previously, it has been
described that synthetic flavonoids and some natural
plant-derived flavonoids seem to inhibit T4 50-DI
activity in vivo and are potent inhibitors of hepatic
deiodinase activity in vitro.
32
Another study reported
that the high consumption of flavonoids including
catechin diminished the enzymatic activity of 50-DI
in vivo as well as in vitro.
33
Serum TSH level
0
1
2
3
4
5
6
ng/ml
Control Gr 1.25% Gr 2.5% Gr 5.0%
Bl 1.25% Bl 2.5% Bl 5.0%
a
a
cd
Figure 9. Effect of green and black tea extracts at
different doses on serum thyroid stimulating hormone
(TSH) level in rats. Each bar denotes mean +SD, n¼8.
One-way analysis of variance (ANOVA) test followed by a
multiple comparison ttest was performed. Significance at
p< 0.05.
a
Control versus other groups;
c
green tea 2.5 g%
versus black tea 2.5 g%;
d
green tea 5.0 g% versus black tea
5.0 g%. Gr ¼Green tea extract treatment and Bl¼Black
tea extract treatment.
Serum T4 level
0
1
2
3
4
5
6
7
μg/dl
Control Gr 1.25% Gr 2.5% Gr 5.0%
Bl 1.25% Bl 2.5% Bl 5.0%
a
a
cd
Figure 8. Effect of green and black tea extracts at
different doses on serum T4 level in rats. Each bar denotes
mean +SD, n¼8. One-way analysis of variance
(ANOVA) test followed by a multiple comparison ttest
was performed. Significance at p< 0.05.
a
Control versus
other groups;
c
green tea 2.5 g% versus black tea 2.5 g%;
d
green tea 5.0 g% versus black tea 5.0 g%. Gr ¼green tea
extract treatment and Bl ¼black tea extract treatment.
Chandra AK et al. 893

It is important to point out that thyroid
gland weight was increased significantly in a
dose-dependent manner after oral administration of
un-fractionated green tea for a period of 30 days,
whereas, weight of thyroid gland increased signifi-
cantly only after high dose of black tea exposure for
same duration. It has been previously reported that
oral administration of diet containing GTE catechins
(P-60) at 5%dose to male rats for 28 weeks induced
goiter.
13,14
In the present study, it was found that oral
administration of green tea (at moderate and high
doses) and black tea (only at high dose) induced
hypertrophy and/or hyperplasia of thyroid gland,
which are common in the thyroid of rats treated with
goitrogenic substances.
34,35
In 2.5 g%group of green
tea, thyroid follicles were diffusely hyperplastic, irre-
gularly shaped, lined with enlarged cuboidal or
columnar epithelium with depleted colloid, whereas
at a dose of 5.0 g%, colloid-rich follicles with flat
epithelial cell with mononuclear cell infiltration were
observed that was similar to the changes observed fea-
tures after un-fractionated black tea exposure at high
dose (5.0 g%). Consistent with those observations,
Sakamoto et al.
13
reported that marked hypertrophy
and hyperplasia of the follicles, some with depletion
of colloid and some with rich colloid after the oral
administration of 5.0 g%GTE catechin (P-60) in rats
in a 13-week study while the changes were less severe
after relatively lower doses.
Another finding in this study is the elevated TSH
and decreased serum T3 and T4 levels. Such altera-
tion in serum thyroid hormone profile by green tea
catechin P-60 has been reported earlier.
13,14
The pres-
ent data suggests that oral administration of green tea
(at moderate and high doses) and black tea (only at
high dose) exert an antithyroid effect, reducing T3
and T4 concentrations, that in turn increase TSH
secretion and consequently resulting hypertrophic/
hyperplastic changes in the thyroid follicles.
The present study indicates that low-dose exposure
to un-fractionated GTE and BTEs would have no sig-
nificant impact on thyroid hormone status. The pres-
ent in vivo study reveals that chronic dietary
administration of the green tea at moderate and high
doses and black tea only at a high dose induce a mor-
phological as well as functional state of hypothyroid-
ism in rats by interfering with the activity of the
enzyme for thyroid hormone synthesis and for thyroi-
dal deiodination of T4 to T3, resulting in decreased
T3 level and augmented TSH level. This study also
demonstrated that the relative adverse effect of green
tea on thyroid physiology is much more than that of
the black tea, probably because green tea contains
about 30%w/w of catechins, whereas black tea con-
tains less tea catechins (3%–10%w/w). This present
in vivo study may propose that the natural flavonoids,
catechin present in green and black tea in different
proportions, have the potential to induce hypothyroid-
ism and goiter in rats if this beverage is consumed rel-
atively at high doses for prolonged duration.
In considering a risk to humans, species difference
should be considered. Many experiments show that
rodents are highly sensitive to goitrogenic agents in
comparison with humans, because rats are lacking
in high-affinity thyroxine-binding globulin which is
present in humans and plasma half-life of T4 in rats
(1224 hours) is shorter than in humans (59
days).
13,14,36,37
This study suggests that oral adminis-
tration of green tea (mainly) and black tea only at high
dose has the potential to induce hypothyroidism and
goiter in rats; however, human tea drinkers are
unlikely to be at risk.
Acknowledgements
The authors also thank Dr Ashu Gulati, Scientist, IHBT,
Palampur (HP), India, for providing green and black tea
samples and for the measurement of concentrations of cate-
chins in green tea samples. The authors also thank Tea
Research Association, Tocklai Experimental Station, Jorhat,
Assam, for providing black tea samples. The authors are
grateful to Prof. Arun K Ray (Bose Institute, India) for his
contribution in conducting enzyme study. The authors thank
Dr Syed N Kabir, Scientist, Cell Biology & Physiology
Division, Indian Institute of Chemical Biology (IICB),
Kolkata, India, for his help in conducting RIA of TSH.
Funding
The National Tea Research Foundation (NTRF; Scheme
code no. NTRF: 106/07), Kolkata, India, has provided
scholarship to Neela De for this work.
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