Chemopreventive efficacy of curcumin and piperine during 7,12-dimethylbenz [a]anthracene-induced hamster buccal pouch carcinogenesis

O r i g i n a l A r t i c l e
Singapore Med J 2009; 50 (2) : 139
Chemopreventive efficacy of curcumin
and piperine during 7,12-dimethylbenz
[a]anthracene-induced hamster buccal
pouch carcinogenesis
Manoharan S, Balakrishnan S, Menon V P, Alias L M, Reena A R
ABSTRACT
Keywords: 7,12-dimethylbenz[a]anthracene,
Introduction: Oral carcinoma accounts for 40–50 antioxidants, buccal pouch carcinogenesis,
percent of all cancers in India. Tobacco chewing, curcumin, lipid peroxidation, oral carcinoma,
smoking and alcohol consumption are the major piperine
risk factors associated with the high incidence of Singapore Med J 2009; 50(2): 139-146
oral cancer in India. Our aim was to investigate

the chemopreventive potential of curcumin and INTRODUCTION
piperine during 7,12-dimethylbenz[a]anthra-
Oral carcinoma is defined as cancer of the mouth and
cene (DMBA) -induced hamster buccal pouch pharynx including cancer of the lips, tongue, floor of the
carcinogenesis.
mouth, palate, gingiva, alveolar mucosa, buccal mucosa,
oropharynx, tonsils, uvula and salivary glands. India has
Methods: Oral squamous cell carcinoma was recorded the highest incidence of oral cancer, where it
developed in the buccal pouch of Syrian golden accounts for 40%–50% of all cancers. The major risk
hamsters, by painting them with 0.5 percent factors for this cancer include the use of tobacco products
DMBA in liquid paraffin, three times a week for 14 (tobacco smoking and chewing) and alcohol, which
weeks. The tumour incidence, tumour volume and accounts for 75% of all oral cancers. Oral cancer most
burden were determined in the buccal pouches. often occurs in people over the age of 40 years, and about
The status of phase II detoxification agents, lipid half of the patients afflicted will die within five years
peroxidation and antioxidants were estimated by of diagnosis.(1,2) The 7,12-dimethylbenz[a]anthracene
specific colorimetric methods.
(DMBA), an organ-specific carcinogen, on metabolic
Department of
activation produces the ultimate carcinogen, dihydrodiol Biochemistry and
Results: We observed 100 percent tumour epoxide, which can mediate neoplastic transformation Biotechnology,
Faculty of Science,
formation in DMBA-alone painted hamsters. by inducing DNA damage, generating excess reactive Annamalai University,
Annamalainagar
Disturbances in the status of lipid peroxidation, oxygen species (ROS) and mediating the chronic 608002,
Tamil Nadu,
antioxidants and phase II detoxification agents inflammatory process. Oral carcinogenesis induced by India
were noticed in DMBA-alone painted hamsters. DMBA closely resembles a human oral tumour, both Manoharan S, MSc,
Oral administration of curcumin (80 mg/kg body histologically and morphologically. DMBA-induced MPhil, PhD
Reader
weight) and piperine (50 mg/kg body weight) experimental oral carcinogenesis can thus be used as
to DMBA-painted hamsters on alternate days
Balakrishnan S, MSc,
an ideal model for studying chemoprevention of oral MPhil, PhD
to DMBA painting for 14 weeks completely cancer.(3,4) Measurement of the activities of detoxification Research Scholar
prevented the formation of oral carcinoma. Also, agents such as glutathione-S-transferase (GST) and Menon VP, MSc, PhD
Professor and Head
curcumin and piperine restored the status of lipid glutathione reductase (GR) in the liver may help to assess
peroxidation, antioxidants and detoxifying agents the chemopreventive potential of the test compound. GST Alias LM, MSc, MPhil
Research Scholar
in DMBA-painted hamsters.
detoxifies carcinogens either by destroying their active
Reena AR, MSc, MPhil
reactive centres or by facilitating their excretion through Research Scholar
Conclusion: The chemopreventive efficacy of a conjugation process. GR, an important enzyme required Correspondence to:
curcumin and piperine is probably due to their for reduced glutathione (GSH) maintenance, catalyses Dr Shanmugam
Manoharan
antilipidperoxidative and antioxidant potential as reduced nicotinamide adenine dinucleotide phosphate Tel: (91) 4144 238 343
Fax: (91) 4144 238 080
well as their modulating effect on the carcinogen (NADPH)-dependent reduction of glutathione disulphide Email: sakshiman@
detoxification process.
rediffmail.com
to GSH.(5)

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Singapore Med J 2009; 50 (2) : 140
Table I. Incidence of oral squamous cell carcinoma in DMBA alone, DMBA + piperine and DMBA + curcumin treated
hamsters.
Groups
Tumour incidence
Total no. of tumours/total
Tumour volume
Tumour burden

(oral squamous cell carcinoma)
no. of animals tested
(mm3)/animals
(mm3)/animals
DMBA alone
100% tumour formation
37/10
378.3 ± 32.1
1,399.7 ± 98.6
DMBA + piperine
No tumour formation
0/10
–
–
DMBA + curcumin
No tumour formation
0/10
–
–
Tumour volume was measured using the formula, ? = , where D1, D2 and D3 are the three diameters (mm) of the
tumour. Tumour burden was calculated by multiplying tumour volume and the number of tumours/animal.
Table II. Histopathological changes in the buccal pouch of hamsters in control and experimental animals in each
group.
Groups
Hyperkeratosis
Hyperplasia
Dysplasia
Squamous cell carcinoma
Control
Not observed
Not observed
Not observed
Not observed
DMBA
Severe
Severe
Severe
Well differentiated
DMBA + piperine
Moderate
Moderate
Mild
–
DMBA + curcumin
Moderate
Moderate
Mild
–
Piperine alone
Not observed
Not observed
Not observed
Not observed
Curcumin alone
Not observed
Not observed
Not observed
Not observed

A large number of chemical carcinogens, including effects on cell proliferation and antioxidant function are
DMBA, mediate carcinogenesis through free radical- considered to be good chemopreventive agents. A large
mediated oxidative damage to cells and tissues. Enormous number of active principles from traditional medicinal
production of ROS due to oxidative stress in the system plants have been reported to have chemopreventive
can induce strand breaks and can modify DNA bases properties.(13,14) Circulatory lipid peroxidation and
contributing to mutagenesis and carcinogenesis.(6) The antioxidant status could be used as biomarkers of
major target of ROS in vivo or in vitro is membrane lipid chemoprevention in experimental carcinogenesis.(15)
peroxidation that leads to abnormalities in the structural
Curcumin, the active principle in Curcuma
integrity and function of the cell membrane.(7) Lipid longa (turmeric), has been shown to have potent
peroxidation, a free radical-mediated chain reaction, has anti-inflammatory, antioxidant and anticarcinogenic
been implicated in the pathogenesis of several disorders properties.(16) Several studies have pointed out that
including oral carcinoma.(8) Increased levels of lipid curcumin has the ability to inhibit carcinogen-DNA adduct
peroxidation byproducts play a role in the early phases formation and the inhibition of tumours in experimental
of tumour growth. An inverse association between lipid carcinogenesis.(17) It has been demonstrated that the
peroxidation and the rate of cell proliferation in tumour topical application of curcumin inhibits DMBA initiation
tissues has been reported.(9) Mammalian cells possess and 12-O-tetradeconoylphorbol 13-acetate promotion
an array of enzymatic (superoxide dismutase [SOD], of skin tumours.(18) Curcumin is a potent scavenger of
catalase [CAT] and glutathione peroxidase [GPx]) and superoxide anion, hydroxyl radical, singlet oxygen, nitric
non-enzymatic (Vitamins C and E, and GSH) defence oxide and peroxinitrite.(16) In vitro, curcumin has been
mechanisms to protect the cells and tissues from the shown to induce the apoptosis of a wide variety of cancer
deleterious effects of free radical-mediated oxidative cells.(19) Piperine, a pungent alkaloid constituent of black
damage. The administration of antioxidants has been and long peppers, possesses diverse pharmacological
shown to have beneficial effects on the regression of actions, such as anti-inflammatory, antimicrobial,
tumours in animals. Anticarcinogenic activity has hepatoprotective and anticancer effects.(20) It has
been demonstrated with vitamin E, vitamin C and been reported that piperine inhibits xenobiotic (drug)
glutathione.(10-12)
metabolising enzymes and enhances cellular antioxidant

Chemoprevention is a promising and novel strategy status.(21) Piperine has been shown to reduce liver lipid
for the prevention, inhibition, suppression and reversal of peroxidation and protect against oxidative damages
carcinogenesis through the use of natural plant products induced by a number of chemical carcinogens.(22) To the
and synthetic agents. It has been suggested that compounds best of our knowledge, we have found no scientific studies
that possess antimutagenic, anticarcinogenic, inhibitory on the chemopreventive efficacy of piperine in DMBA-

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Singapore Med J 2009; 50 (2) : 141
Fig. 1 Photograph shows the gross appearance of oral squamous Fig. 2 Photomicrograph shows hyperkeratosis ( ), hyper-
cell carcinoma ( ) noticed in DMBA-alone painted plasia ( ), dysplasia ( ) and well-differentiated squamous
animals.
cell carcinoma with keratin pearls ( ) observed in the
buccal pouch of DMBA-alone painted hamsters (Haematoxylin
& eosin, × 10).
induced hamster buccal pouch carcinogenesis. Although of sacrifice. The total number of tumours was counted
the cancer chemopreventive potential of curcumin macroscopically by an oral pathologist (CRR).
and piperine has been repeatedly reported in various
The institutional animal ethics committee (Reg.
experimental carcinogenesis, the biochemical mechanistic no.160/1999/CPCSEA), Annamalai University,
pathway for their chemopreventive effects has not been Annamalainagar, India, approved the experimental
reported in DMBA-induced oral carcinogenesis. Thus, the design. The animals were maintained as per the principles
present study was undertaken to find out the biochemical and guidelines of the ethical committee for animal care of
mechanistic pathway for the chemopreventive efficacy Annamalai University in accordance with Indian National
of curcumin and piperine during DMBA-induced oral Law on animal care and use. A total of 60 hamsters were
carcinogenesis.
randomised into six groups with ten animals in each
group. Group I served as the control group, where the
METHODS
animals were painted with liquid paraffin (vehicle) alone
DMBA, curcumin and piperine were obtained from three times a week for 14 weeks on their left buccal
Sigma-Aldrich Chemical Pvt Ltd, Bangalore, India. All pouches. Groups II–IV animals were painted with 0.5%
other chemicals used were of analytical grade, purchased DMBA in liquid paraffin three times a week for 14 weeks
from Himedia Laboratories, Mumbai, India. Male golden on their left buccal pouches. Group II animals received
Syrian hamsters, aged 8–10 weeks, weighing 80–120g, no other treatment. Groups III and IV animals were orally
were purchased from the National Institute of Nutrition, administered with piperine (50 mg/kg body weight) and
Hyderabad, India and were maintained in the Central curcumin (80 mg/kg body weight) respectively, starting
Animal House, Rajah Muthaiah Medical College and one week before exposure to the carcinogen and continued
Hospital, Annamalai University. The animals were on alternate days to DMBA painting until the animals
housed five in a polypropylene cage and provided with a were sacrificed. Groups V and VI animals received oral
standard pellet diet (Agro Corporation Pvt Ltd, Bangalore, administration of piperine (50 mg/kg body weight) and
India) and water ad libitum. The standard pellet diet is curcumin (80 mg/kg body weight) alone, respectively,
composed of 21% protein, 5% lipids, 4% crude fibre, 8% throughout the experimental period. The experiment
ash, 1% calcium, 0.6% phosphorus, 3.4% glucose, 2% was terminated at the end of 14 weeks and all animals
vitamin and 55% nitrogen-free extract (carbohydrates). were sacrificed by cervical dislocation. Biochemical
The animals were maintained under controlled conditions studies were conducted on the blood and buccal mucosa
of temperature (27 ± 2°C) and humidity (55 ± 5%) with of control and experimental animals in each group. For
a 12 h light/dark cycle. Tumours were induced in each histopathological examination, buccal mucosa tissues
hamster’s buccal pouch through the topical application of were fixed in 10% formalin and routinely processed and
0.5% DMBA in liquid paraffin three times a week for 14 embedded with paraffin, 2–3 ?m sections were cut in a
weeks.(4,10) The total number of tumours in the hamster’s rotary microtome and stained with haematoxylin and
buccal pouch was determined macroscopically at the time eosin.

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Singapore Med J 2009; 50 (2) : 142
a known volume using 0.2 M isotonic Tris-HCl buffer
3a
GSH
(pH 7.4). Aliquots from these preparations were used for
the estimation of thiobarbituric acid reactive substances
(TBARS) and vitamin E.

tissue

Tissue samples from the animals were washed with
ice cold saline and homogenised using an appropriate
nmol/mg
buffer (GST: 0.3 M phosphate buffer, pH 6.5; GR: 0.1
M phosphate buffer, pH 7.4; TBARS: 0.025 M Tris-
HCl buffer, pH 7.5; GSH and GPx: 0.4 M phosphate
Groups
buffer, pH 7.0; SOD: 0.025 M sodium pyrophosphate
3b
GST
buffer, pH 8.3; CAT: 0.01 M phosphate buffer, pH 7.0)
in an all-glass homogeniser with a teflon pestle and used
otein
for biochemical estimations. The activity of GST in

pr

conjugate
liver tissue homogenate was assayed using the method
employed by Habig et al.(25) GST activity was measured

CDNB
by incubating the tissue homogenate with the substrate 1-
nmol
f
ormed/min/mg
chloro-2,4-dinitrobenzene. The absorbance was followed
for 5 min at 540 nm after the reaction was started by the
Groups
addition of GSH. GR activity in liver tissue homogenate
3c
GR
was assayed using the method employed by Carlberg
and Mannervik.(26) The enzyme activity was assayed
by measuring the formation of GSH when the oxidised

o
xidised
otein
glutathione is reduced by NADPH.

pr

Lipid peroxidation was estimated as evidenced by the
formation of TBARS. TBARS in plasma were assayed by

NADPH

min/mg

of
the method described by Yagi.(27) Plasma was deproteinised
per
nmol
with phosphotungstic acid and the precipitate was treated
Groups
with thiobarbituric acid at 90°C for an hour. The pink
Fig. 3 Bar charts show the activities of detoxification agents, (a) colour formed gave a measure of the TBARS, which was
GSH; (b) GST; and (c) GR, in liver homogenate of control and read at 530 nm. TBARS in erythrocytes and erythrocyte
experimental animals in each group (n = 10).
Groups I: Control; II: DMBA; III: DMBA + piperine; IV: DMBA + membrane was estimated using the method employed by
curcumin; V: Piperine alone; VI: Curcumin alone. Values are ex-
Donnan.(28) The pink chromogen formed by the reaction
pressed as mean ± SD for ten hamsters in each group. Values of thiobarbituric acid with breakdown products of lipid
that do not share a common superscript in the same graph differ
significantly at p < 0.05 (DMRT).
peroxides was read at 535 nm. Tissue lipid peroxidation
was done using the method employed by Ohkawa et
al.(29) The colour formed by the reaction of thiobarbituric

Blood samples were collected into heparinised tubes. acid with breakdown products of lipid peroxidation was
Plasma was separated by centrifugation at 1,000 g for 15 measured colorimetrically at 532 nm. The GSH levels
min. The buffy coat was removed and the packed cells in the plasma, erythrocytes, liver and buccal mucosa
were washed three times with physiological saline. The were determined by the method described by Beutler and
erythrocyte membrane was prepared using the method Kelley.(30) The technique involved protein precipitation by
employed by Dodge et al(23) and modified by Quist.(24) The metaphosphoric acid and spectrophotometric assay at 412
erythrocytes remaining after the removal of plasma were nm of the yellow derivative obtained by the reaction of
washed three times with 310 mM isotonic Tris-HCl buffer the supernatant with 5-5’-dithiobis-2-nitrobenzoic acid.
(pH 7.4). Haemolysis was carried out by pipetting out
The level of plasma vitamin C was determined by
the washed erythrocyte suspension into polypropylene the method described by Omaye et al.(31) The dehydro-
centrifuge tubes, which contained 20 mM hypotonic ascorbic acid formed from the oxidation of vitamin C by
Tris-HCl buffer (pH 7.2). The erythrocyte membranes copper, formed a coloured product on treatment with 2,4-
were sedimented in a high-speed cooling centrifuge dinitrophenylhydrazine, whose absorbance was measured
at 20,000 g for 40 min. The supernatant was decanted at 520 nm.
and the erythrocyte membrane pellet was made up to
The vitamin E level in the plasma and erythrocyte

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Singapore Med J 2009; 50 (2) : 143
Table III. Status of plasma TBARS and antioxidants in control and experimental animals in each group.
Groups
TBARS
GSH
Vitamin C
Vitamin E
SOD
CAT
GPx

(nmoles/ml)
(mg/dL)
(mg/dL)
(mg/dL)
(UA/ml)
(UB/ml)
(UC/L)
Control
2.02 ± 0.18a
27.3 ± 1.8a
1.34 ± 0.08a
1.38 ± 0.12a
2.61 ± 0.23a
0.93 ± 0.07a
109.7 ± 9.1a
DMBA
4.78 ± 0.42b
17.2 ± 2.3b
0.72 ± 0.09b
0.74 ± 0.13b
1.69 ± 0.19b
0.51 ± 0.09b
76.6 ± 9.3b
DMBA + piperine
2.32 ± 0.23c
24.3 ± 2.2c
1.18 ± 0.11c
1.20 ± 0.09c
2.32 ± 0.20c
0.80 ± 0.08c
94.2 ± 8.5c
DMBA + curcumin
2.27 ± 0.19c
25.1 ± 1.7c
1.22 ± 0.09c
1.24 ± 0.10c
2.37 ± 0.18c
0.84 ± 0.07c
98.6 ± 8.7c
Piperine alone
2.02 ± 0.17a
27.5 ± 1.6a
1.35 ± 0.12a
1.37 ± 0.09a
2.62 ± 0.17a
0.93 ± 0.05a
108.9 ± 8.3a
Curcumin alone
2.01 ± 0.14a
27.6 ± 1.7a
1.36 ± 0.07a
1.38 ± 0.07a
2.62 ± 0.21a
0.94 ± 0.07a
109.5 ± 8.9a
Values are expressed as mean ± SD for ten hamsters in each group. Values that do not share a common superscript in the same
column differ significantly at p < 0.05 (DMRT).


A The amount of enzyme required to inhibit 50% NBT reduction.
B Micromoles of hydrogen peroxide utilised/sec.
C Micromoles of glutathione utilised/min.
membranes were determined colorimetrically using in Table I. We observed oral squamous cell carcinoma
the method described by Desai.(32) Vitamin E present in formation in all the hamsters’ buccal pouches painted
the lipid residue forms a pink-coloured complex with with DMBA alone for 14 weeks; therefore the tumour
bathophenanthroline-phosphoric acid reagent, which incidence was 100%. The total number of oral
was measured at 536 nm. Tissue vitamin E was measured tumours in the buccal pouches was counted and the
using the fluorimetric method described by Palan et diameter of each tumour was measured with a vernier
al.(33) The lipid extracts were dried under nitrogen and caliper. The tumour volume was calculated by the
the residues were suspended in 66% ethanol, followed formula, ? = , where D1, D2 and D3 are
by the addition of 4 ml of hexane and 0.6 ml of 60% the three diameters (mm) of the tumours. Tumour burden
H2SO4. The fluorescence intensity of vitamin E extracted was calculated by multiplying tumour volume and the
to the hexane layer was measured at an excitation of 295 number of tumours/hamster. The mean tumour volume
nm and emission of 320 nm. SOD activity was assayed and burden of tumour-bearing hamsters (Group II) were
using the method employed by Kakkar et al,(34) based on found to be 378.3 mm3 and 1,399.7 mm3, respectively.
the 50% inhibition of NADH-phenazine methosulphate Oral administration of piperine (50 mg/kg body weight)
nitroblue tetrazolium (NBT) formation. The colour and curcumin (80 mg/kg body weight) to DMBA-painted
developed was read at 520 nm. One unit of enzyme is hamsters (Groups III and IV, respectively) on alternate
taken as the amount of enzyme required to give 50% days to DMBA painting for 14 weeks completely
inhibition of NBT reduction. The activity of CAT was prevented the formation of oral squamous cell carcinoma.
assayed using the method described by Sinha,(35) based The gross appearance of oral squamous cell carcinoma
on the utilisation of hydrogen peroxide by the enzyme. noticed in DMBA-alone painted hamsters is shown in
The colour developed was read at 620 nm. One unit of Fig. 1.
the enzyme is expressed as ?moles of hydrogen peroxide
The histopathological features observed in the
utilised per minute. The activity of GPx was determined buccal mucosa of the control and experimental animals
using the method employed by Rotruck et al,(36) based in each group are shown in Table II. We observed
on the utilisation of GSH by the enzyme. One unit of severe hyperkeratosis, hyperplasia, dysplasia and well-
the enzyme is expressed as ?moles of GSH utilised differentiated squamous cell carcinoma in the buccal
per minute. The data is expressed as mean ± standard pouches of DMBA-alone painted hamsters (Fig. 2).
deviation (SD). Statistical comparisons were performed Although well-differentiated squamous cell carcinoma
by one-way analysis of variance followed by Duncan’s was not developed in the buccal pouches of DMBA +
multiple range test (DMRT). The results were considered piperine (Group III) and DMBA + curcumin (Group
statistically significant if the p-values were less than IV) treated hamsters, hyperplasia, hyperkeratosis and
0.05.
dysplasia were observed. Control animals treated with
piperine and curcumin showed well-defined and intact
RESULTS
epithelial layers similar to that of the control animals.
The tumour incidence, tumour volume and tumour burden The severity of pathological changes was determined
in DMBA-painted animals, DMBA + piperine treated by the oral pathologist (CRR), when examining the
animals and DMBA + curcumin treated animals are shown histopathological slides under the microscope.

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Singapore Med J 2009; 50 (2) : 144
Table IV. TBARS and antioxidants status in erythrocytes of control and experimental animals in each group.
Groups
Erythrocyte
Erythrocyte
Vitamin E
Erythrocytes Erythrocyte
Erythrocyte
Erythrocyte

TBARS
membrane
(?g/mg
GSH (mg/dL) lysate SOD
lysate CAT
lysate GPx

(pmoles/
TBARS
protein)

(UA/mg Hb)
(UB/mg Hb)
(UC/g Hb)

mg Hb) (nmoles/


mg protein)
Control
1.48 ± 0.12a
0.32 ± 0.03a
2.56 ± 0.16a
50.3 ± 4.1a
2.50 ± 0.17a
1.36 ± 0.08a
15.62 ± 1.30a
DMBA
2.85 ± 0.33b
1.12 ± 0.12b
1.71 ± 0.19b
31.4 ± 2.9b
1.52 ± 0.19b
0.64 ± 0.09b
8.21 ± 0.93b
DMBA + piperine
1.68 ± 0.13c
0.40 ± 0.06c
2.23 ± 0.23c
44.6 ± 3.9c
2.19 ± 0.21c
1.24 ± 0.07c
13.34 ± 1.28c
DMBA + curcumin
1.61 ± 0.11c
0.38 ± 0.05c
2.28 ± 0.26c
45.7 ± 4.3c
2.24 ± 0.29c
1.26 ± 0.08c
13.92 ± 1.31c
Piperine alone
1.49 ± 0.15a
0.32 ± 0.08a
2.57 ± 0 21a
51.1 ± 4.6a
2.51 ± 0.15a
1.37 ± 0.08a
15.61 ± 1.40a
Curcumin alone
1.47 ± 0.12a
0.31 ± 0.05a
2.58 ± 0.17a
51.7 ± 4.1a
2.52 ± 0.18a
1.37 ± 0.09a
15.73 ± 1.35a
Values are expressed as mean ± SD for ten hamsters in each group. Values that do not share a common superscript in the same
column differ significantly at p < 0.05 (DMRT).


A The amount of enzyme required to inhibit 50% NBT reduction.
B Micromoles of hydrogen peroxide utilised/sec.
C Micromoles of glutathione utilised/min.

The activities of phase II detoxification enzymes as compared to control animals. Oral administration
(GST and GR) and GSH level in the livers of control of piperine and curcumin to DMBA-painted animals
and experimental animals in each group are shown in (Groups III and IV, respectively) brought back the
Fig. 3. The activities of liver detoxification agents were concentration of TBARS and antioxidants to near normal
significantly decreased in tumour-bearing hamsters range. Hamsters treated with piperine and curcumin alone
(Group II), as compared to control hamsters. Oral (Groups V and VI, respectively) showed no significant
administration of piperine (50 mg/kg body weight) and difference in TBARS and antioxidants status as compared
curcumin (80 mg/kg body weight) to DMBA-painted to control hamsters (Group I).
hamsters (Groups III and IV, respectively) significantly
brought back the activities of detoxification agents to DISCUSSION
near normal status. Hamsters treated with piperine and In the present study, we have investigated the
curcumin alone (Groups V and VI, respectively) showed chemopreventive efficacy of curcumin and piperine in
no significant difference in the status of detoxification DMBA-induced hamster buccal pouch carcinogenesis.
agents as compared to control hamsters.
The chemopreventive efficacy was assessed by

The status of TBARS and antioxidants (Vitamins monitoring the percentage of tumour-bearing animals,
E and C, GSH, SOD, CAT and GPx) in plasma and the tumour volume and burden as well as by analysing
erythrocytes of control and experimental animals in the status of detoxification agents, lipid peroxidation
each group are respectively shown in Tables III and IV. and antioxidants in DMBA-painted animals. Hamsters
The concentration of TBARS was increased, whereas painted with 0.5% DMBA for 14 weeks in their buccal
the status of antioxidants was significantly decreased in pouches developed well-differentiated squamous cell
tumour-bearing animals (Group II) as compared to control carcinoma. Oral administration of piperine and curcumin
animals. Oral administration of piperine and curcumin to completely prevented the tumour formation in DMBA-
DMBA-painted animals (Groups III and IV, respectively) painted hamsters (Groups III and IV). Our results thus
significantly brought back the concentrations of TBARS suggest that curcumin and piperine may act as either
and antioxidants to near normal status. Hamsters treated antiproliferators or antipromotors during DMBA-induced
with piperine and curcumin alone (Groups V and VI, oral carcinogenesis.
respectively) showed no significant difference in TBARS
Chemical carcinogens can bind to DNA and result
and antioxidants status as compared to control hamsters in mutagenic events that contribute to malignant
(Group I).
transformation.(37) A number of studies have reported

The status of TBARS and antioxidants in the buccal that chemopreventitive agents convert DNA-damaging
mucosa of the control and experimental animals in each entities into excretable metabolites by stimulating the
group is shown in Table V. A decrease in the TBARS level action of detoxification agents such as GST.(5,38) In the
and disturbances in antioxidants status (vitamin E, GSH present study, the activities of GST, GR and GSH levels
and GPx were increased; SOD and CAT were decreased) were decreased in DMBA-painted animals as compared
were noticed in tumour-bearing animals (Group II) to control animals. Oral administration of piperine and

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Singapore Med J 2009; 50 (2) : 145
Table V. Buccal mucosa TBARS and antioxidants status in control and experimental animals in each group.
Groups
TBARS (nmoles/
GSH (mg/
Vitamin E (mg/
SOD (UA/mg
CAT (UB/mg
GPx (UC/g

100 mg
100 mg
100 mg
protein)
protein)
protein)

protein)
tissues)
tissues)
Control
69.3 ± 5.8a
6.01 ± 0.48a
1.64 ± 0.15a
4.91 ± 0.36a
36.8 ± 2.9a
6.48 ± 0.42a
DMBA
39.8 ± 4.1b
12.3 ± 1.30b
2.91 ± 0.32b
2.98 ± 0.31b
20.1 ± 2.6b
12.7 ± 1.20b
DMBA + piperine
59.8 ± 4.9c
7.08 ± 0.98c
1.98 ± 0.23c
4.32 ± 0.35c
31.8 ± 3.4c
7. 49 ± 0.69c
DMBA + curcumin
62.2 ± 4.6c
6.84 ± 0.82c
1.92 ± 0.23c
4.36 ± 0.36c
33.1 ± 3.1c
7.41 ± 0.72c
Piperine alone
69.2 ± 5.9a
6.03 ± 0.42a
1.65 ± 0.14a
4.95 ± 0.38a
37.1 ± 2.8a
6.52 ± 0.48a
Curcumin alone
68.8 ± 5.7a
6.04 ± 0.41a
1.67 ± 0.13a
4.99 ± 0.46a
37.6 ± 3.2a
6.58 ± 0.51a
Values are expressed as mean ± SD for ten hamsters in each group. Values that do not share a common superscript in the same
column differ significantly at p < 0.05 (DMRT).


A The amount of enzyme required to inhibit 50% NBT reduction.
B Micromoles of hydrogen peroxide utilised/sec.
C Micromoles of glutathione utilised/min.
curcumin to DMBA-painted animals restored the activities in tumour tissues.(40) Thus, the decreased susceptibility
of GST, GR, GSH and glutathione, which indicated their of tumour tissues to lipid peroxidation can be related to
role in carcinogen detoxification. The chemopreventive increased cel proliferation occurring in oral carcinogenesis
effect of piperine and curcumin is probably due to or the poor availability of PUFA in tumour tissues.
their inhibitory role on metabolic activation of DMBA Glutathione and GPx have regulatory effects on cellular
by stimulating the activities of phase II detoxification proliferation and are over-expressed in various malignant
agents.
tumours.(7,41) An increased level of vitamin E has been

The measurement of serum or plasma lipid reported in tumour tissues of several cancers, including oral
peroxidation byproducts helps in assessing the extent of carcinoma.(4,10) Lowered levels of tumour tissue TBARS
tissue damage.(8) It has been suggested that excessively also indicate that tumour tissues have sequestered these
generated lipid peroxides at the primary site could be antioxidants from circulation to enhance their antioxidant
transferred through circulation to other organs and provoke capacity as well as to meet their nutrient requirements.
damage by propagating lipid peroxidation.(8) Erythrocytes Tumour tissues produce substantial amounts of
are highly susceptible to ROS-mediated oxidative damage superoxides and hydrogen peroxides.(39) Lowered activities
in pathological conditions.(7) Increased plasma TBARS of SOD and CAT in tumour tissues are probably due to the
observed in tumour-bearing hamsters is probably due to exhaustion of these enzymes to scavenge overproduced
the overproduction and diffusion of lipid peroxidation superoxides and hydrogen peroxides in tumour cells. Oral
byproducts from the damaged tissues with consequent administration of piperine and curcumin restored the status
leakage into plasma. Non-enzymatic and enzymatic of lipid peroxidation and antioxidants in circulation and
antioxidants play a protective role against neoplastic the buccal mucosa tissues of DMBA-painted animals. Our
transformation and ROS-mediated oxidative stress.(4,7) results thus suggest that piperine and curcumin displayed a
Tumour cells sequester essential nutrients from circulation potent antioxidant function during oral carcinogenesis.
to meet the demands of a growing tumour.(4) Decreased
The present study thus demonstrated the
levels of vitamins E and C and GSH content in plasma chemopreventive efficacy of curcumin and piperine in
and erythrocytes are probably due to their utilisation by DMBA-induced hamster buccal pouch carcinogenesis. The
tumour tissues to meet their nutrient demands for growth. chemopreventive potential of curcumin and piperine are
Lowered activities of SOD, catalase and GPx in plasma and probably due to their antilipidperoxidative and antioxidant
erythrocytes are probably due to the exhaustion of these potential or modulating effect on the carcinogen
enzymes to scavenge excessively-generated ROS in the detoxification process. Further studies are, however,
system.
required in order to better understand the underlying

Tumours have shown less susceptibility to lipid mechanisms of the chemopreventive actions of curcumin
peroxidation as opposed to normal cells. An inverse and piperine.
association between lipid peroxidation and the rate of
cell division in tumours has been suggested.(9,39) The ACKNOWLEDGEMENTS
poor availability of peroxidative substrates such as Financial support from the University Grants Commission,
polyunsaturated fatty acids (PUFA) has been demonstrated New Delhi is gratefully acknowledged. Mr S Balakrishnan

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Singapore Med J 2009; 50 (2) : 146
was working as a Project Associate under this scheme. 19. Marin YE, Wall BA, Wang S, et al. Curcumin downregulates the
We are also indebted to Dr CR Ramachandran, Faculty of
constitutive activity of NF-kappaB and induces apoptosis in novel
mouse melanoma cells. Melanoma Res 2007; 17:274-83.
Dentistry, Rajah Muthaiah Dental College and Hospital, 20. Cole OF. Pharmacological studies of piperine; I. Effects of piperine
Annamalai University, for his invaluable assistance with
on transmural nerve stimulation. Planta Med 1985; 51:153-6.
the pathological work.
21. Selvendiran K, Banu SM, Sakthisekaran D. Oral supplementation
of piperine leads to altered phase II enzymes and reduced DNA
damage and DNA-protein cross links in Benzo[a]pyrene induced
REFERENCES
experimental lung carcinogenesis. Mol Cell Biochem 2005;
1. Sankaranarayanan R, Dinshaw K, Nene BM, et al. Cervical and
268:141-7.
oral cancer screening in India. J Med Screen 2006; 13 Suppl 1:
22. Koul IB, Kapil A. Evaluation of the liver protective potential of
S35-8.
piperine, an active principle of black and long peppers. Planta
2. Subapriya R, Thangavelu A, Mathavan B, Ramachandran CR,
Med 1993; 59:413-7.
Nagini S. Assessment of risk factors for oral squamous cell 23. Dodge JT, Mitchell C, Hanahan DJ. The preparation and chemical
carcinoma in Chidambaram, Southern India: a case-control study.
characteristics of haemoglobin-free ghosts of human erythrocytes.
Eur J Cancer Prev 2007; 16:251-6.
Arch Biochem Biophys 1963; 100:119-30.
3. Miyata M, Furukawa M, Takahasi K, Gonzalez FJ, Yamazoe 24. Quist EE. Regulation of erythrocyte membrane shape by Ca2+.
Y. Mechanism of 7,12-dimethylbenz[a]anthracene-induced
Biochem Biophys Res Commun 1980; 92:631-7.
immunotoxicity: role of metabolic activation at the target organ. 25. Habig WH, Pabst MJ, Jakoby WB. Glutathione-S-transferase. The
Jpn J Pharmacol 2001; 86:302-9.
first enzymatic step in mercapturic acid formation. J Biol Chem
4. Manoharan S, Kavitha K, Senthil N, Renju GL. Evaluation of
1974; 249:7130-9.
anticarcinogenic effects of Clerodendron inerme on 7,12-dimethyl
26. Carlberg I, Mannervik B. Glutathione reductase. Methods
benz(a)anthracene-induced hamster buccal pouch carcinogenesis.
Enzymol 1985; 113:484-90.
Singapore Med J 2006; 47:1038-43.
27. Yagi K. Lipid peroxides and human diseases. Chem Phys Lipids
5. Wilkinson J 4th, Clapper ML. Detoxification enzymes and
1987; 45:337-51.
chemoprevention. Proc Soc Exp Biol Med 1997; 216:192-200.
28. Donnan SK. The thioharbituric acid test applied to tissues from
6. Okada F. Beyond foreign-body-induced carcinogenesis: impact
rats treated in various ways. J Biol Chem 1950; 182:415-9.
of reactive oxygen species derived from inflammatory cells in 29. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal
tumorigenic conversion and tumor progression. Int J Cancer 2007;
tissues by thiobarbituric acid reaction. Anal Biochem 1979;
121:2364-72.
95:351-8.
7. Kolanjiappan K, Manoharan S, Kayalvizhi M. Measurement of 30. Beutler E, Kelly BM. The effect of sodium nitrite on red cell GSH.
erythrocytes lipids, lipid peroxidation, antioxidants and osmotic
Experientia 1963; 19:96-7.
fragility in cervical cancer patients. Clin Chim Acta 2002; 31. Omaye ST, Turnbull JD, Sauberlich&nb