Download rajiv gandhi university of health sciences

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECT DISSERTATION.
1.
NAME OF THE CANDIDATE AND
ADDRESS
SUNIL KUMAR SINGH
DEPT. OF PHARMACOLOGY
SET’s COLLEGE OF PHARMACY
S.R.NAGAR,
DHARWAD-580002
2.
NAME OF THE INSTITUTION
SET’s COLLEGE OF PHARMACY,
S.R.NAGAR
DHARWAD-580002
3.
COURSE OF STUDY AND SUBJECT.
MASTER OF PHARMACY IN
PHARMACOLOGY
4.
DATE OF ADMISSION OF COURSE
JUNE -2007
5.
TITLE OF THE TOPIC
ASSESSMENT OF ANTICARCINOGENECITY AND ANTIMUTAGENICITY
OF LYCOPENE IN SWISS ALBINO MICE.
6.0 BRIEF RESUME OF INTENDDED WORK:
6.1 Need for the study
Cancer is the second largest killer disease in the developed countries. It is
estimated that cancer accounts for more than 20% of the deaths in United States. Based
on the current rate of incidence, it is believed that one in every three person will
develop cancer at sometime during his life1.
Presently no satisfactory treatments of cancer are available. However unified
treatment concepts such as surgery, radiotherapy and chemotherapy often remain the
methods of choice in the treatment of cancer. A major hurdle in treating cancer patients
using chemotherapy is the severe side effects. Radiotherapy not only kills cancer cells
but also normal cells by damaging DNA through production of reactive oxygen species
(ROS). Further, leads to genomic damage in the stem cells.
In recent times, herbs are effectively used in cancer chemotherapy. Plant active
constituents like Vincristine, Vinblastine and Vindesine have been found to possess
profound anticarcinogenic effect2.
Oxidative stress induced by ROS is one of the main foci of recent research related
to cancer and cardiovascular disease. ROS are highly reactive oxidant molecules that
are generated endogenously through regular metabolic activity, lifestyle activity and
diet. They react with cellular components, causing oxidative damage to such critical
cellular biomolecules as lipids, proteins and DNA. There is strong evidence that this
damage may play a significant role in the causation of several chronic diseases3.
Antioxidants are protective agents that inactivate reactive oxygen species and
therefore significantly delay or prevent oxidative damage. Antioxidants such as
superoxide dismutase, catalase and glutathione peroxidase are naturally present within
human cells. In addition, antioxidants such as vitamin E, vitamin C, polyphenols and
carotenoids are available from food. Current dietary guidelines to combat chronic
diseases, including cancer and coronary artery disease, recommend increased intake of
plant foods, including fruits and vegetables, which are rich sources of antioxidants.3,4
The role of dietary antioxidants, including vitamin C, vitamin E, carotenoids and
polyphenols,
In disease, prevention has received much attention in recent years. These antioxidants
appear to have a wide range of anticancer and antiatherogenic properties. These
observations may explain the epidemiological data indicating that diets rich in fruits
and vegetables are associated with a reduced risk of numerous chronic diseases.
Another dietary antioxidant thought to be important in the defense against
oxidation is lycopene (a carotinoid), of which tomatoes are an important dietary source.
Lycopene is a natural pigment synthesized by plants and microorganisms but not by
animals. It is a red, fat-soluble pigment serves as an accessory light-gathering pigment
and protects these organisms against the toxic effects of oxygen and light1.
Lycopene has been found to inhibit proliferation of several types of human
cancer cells, including endometrial, breast, and lung5-7. In addition, in vivo studies have
shown lycopene has tumor-suppressive activity8. Other studies support the hypothesis
that carotenoid-containing plant products, such as lycopene, exert a cancer protective
effect via a decrease in oxidative and other damage to DNA in humans9. Lycopene has
also recently been shown to elevate levels of hepatic reduced glutathione and
biotransformation enzymes, potentially playing a key role in preventing cancer
development at extrahepatic sites. Frequent tomato or lycopene intake was associated
with a reduced risk of prostate cancer3.
Lycopene is a potent antioxidant in vitro and in vivo studies, reducing the
susceptibility of lymphocyte DNA to oxidative damage, inactivating hydrogen
peroxide and nitrogen dioxide, and protecting lymphocytes from nitrogen oxide
induced membrane damage and cell death twice as efficiently as beta-carotene10.
Hence the present study has been designed to investigate the anticarcinogenicity
(Skin carcinogenicit) and anti mutagenicity of Lycopene in Swiss albino mice.
6.2 Review of literature:

Agrawal S and Rao AV3 have studied the beneficial effects of lycopene on
human health and chronic disease.

Agrawal S and Rao AV11 have also studied the effects of lycopene on low
density lipoprotein oxidation.

Arab L12,13 et al. have studied the effect of lycopene in cardiovascular
disease.

Bohm Y14 et al. have studied the Intestinal absorption of lycopene from
different matrices and interactions to other cariotenoids, the lipid status and
the antioxidant capacity of human plasma.

Boileau AC15 et al. have studied the Cis lycopene is more bioavilable than
trans – lycopene in vitro and in-vivo lymph cannulated ferrets.

Clinton SK16 et al cis trans lycopene isomers, have studied the corotenoids
and retinol in the human prostate cancer.

Clinton SK17 have studied lycopene chemistry, biology and implication for
human health and disease.

Heber D and Lu QY18 have studied overview of mechanism of action of
lycopene.

Agrawal RC, Kumar S19 have studied the Preventive of cyclophophamide
induced micronuclear formation in mouse bone marrow by Indole -3carbinol.

Bohm F20 et al .have studied Carotenoids protect against cell membrane
damage by the nitrogendioxide radical .
6.3 OBJECTIVE OF THE STUDY:
The objectives of the proposed study are:1. To study the preventive effect of lycopene using skin carcinogenesis protocol
on Swiss Albino mice.
2. To study the preventive effects of lycopene using bone marrow micronucleus
test.
3. To study the preventive effect of lycopene using bone marrow chromosomal
aberration test.
4. To study the antibacterial effect of lycopene in different strains of Bacteria,
(Diplobacillus, Micrococcus).
7.0 MATERIALS AND METHODS
7.1 ANIMALS :
Swiss Albino mice body weight 15-20 gm will be used for the experiment
7.2 MATERIALS (DRUGS & CHEMICALS):
 Lycopene
 DMBA (7,12-dimethylbenzanthracene )
 Cyclophosphamide.
 Cisplatine.
METHODOLOGY:
1. DMBA (7,12-Dimethylbenzantharacene) induced skin Carcinogenicity 21
Swiss albino mice of body weight 15-20 gm will be taken for this study. The animals
are randomly divided into 8 groups.
Group-I : Untreated control
Group-II: Vehicle control
Group-III: DMBA
Group-IV: DMBA+ Croton oil.
Group-V: DMBA +Croton oil+ lycopene
Group-VI: Lycopene alon.
Group-VII: Croton oil alon.
Group-VIII: Lycopene +DNBA+ Croton oil
Single dose DMBA (0.2ml/kg) will be applied on the skin to the respective groups of
mice. Then croton oil (0.2 ml/kg) will be applied weakly twice for up to 3 months to
the respective groups of mice. Then lycopene will be applied to respective group of
mice half an hour before croton oil application. The following parameters will be
assessed.
Parameters:
 Tumor size
 Histopathology
2. Cyclophasphamide induced Mutagenicity :
Swiss Albino mice (body weight 15-20 gm) will be used.
The animals will be randomly divided into 4 groups
Group-I : Cyclophosphamide control
Group-II: Drug + Cyclophosphamide
Group-III: Drug control
Group-IV: Vehicle control
Single dose of cyclophosphamide will be given intraperitoneally. After 24 hr, drug will
be given to respective group of mice. Further, 48hr post cyclophosphamide treatment,
colchicine will be given i.p. and immediately mice will be dissected. Bone marrow will
be removed and the following parameters will be assessed.
Parameters:
 Bone marrow micronucleus test.
 Bone marrow chromosomal aberration test.
3. Cisplatin induced Mutagenicity:
Animal: Swiss Albino mice body weight 15-20 gm
The animals will be randomly divided into 4 groups
Group-I : Cisplatin control
Group-II: Drug + Cisplatin
Group-III: Drug control
Group-IV: Vehicle control
Single dose of cisplatin will be given intraperitoneally. After 24 hr, drug will be given
to respective group of mice. Further, 48hr post cisplatin treatment, colchicine will be
given i.p. and immediately mice will be dissected. Bone marrow will be removed and
the following parameters will be assessed.
Parameters:
 Bone marrow micronucleus test.
 Bone marrow chromosomal aberration test.
Statistical analysis:
The values will be expressed as mean ± SEM. The data will be analyzed by using one
way ANOVA followed by Tukey’s post hoc test.
7.3 MEHTOD OF COLLECTION OF DATA:
Experimental data will be collected by subjecting Swiss Albino mice to various
studies.
7.4 Dose the study requires any investigations or intervention to be conducted on
patients or other humans or animals? If so, please describe briefly.
The above study require in vivo and in vitro screening techniques on Swiss
Albino mice.
7.5 Has ethical clearance been obtained from your institution in case of 7.3?
The copy of the ethical clearance certificate is enclosed.
8.0 LIST OF REFERENCES:
1. Gartner C, Stahl W, Sies H. Lycopene is more bioavailable from tomato paste than
from fresh tomatoes. Am J Clin Nutr 1997;66:116-22.
2. Satyanarayana U. Biochemistry. India: Books and allied (P) Ltd; 2004.
3. Agarwal S, Rao AV. Tomato lycopene and its role in human health and chronic
diseases. CMAJ 2000;163:739-44.
4. Bohm F,Tinkeler JH, Truscott TG. Carotenoids protect against cell membrane
damage by the nitrogendioxide radical. Nat Med 1995;1:98-9.
5. Levy J, Bosin E, Feldman B, et al. Lycopene is a more potent inhibitor of human
cancer cell proliferation than either alpha-carotene or beta-carotene. Nutr
Cancer1995;24:257-266.
6. Amir H, Karas M, Giat J, et al. Lycopene and1,25-dihydroxyvitamin D3 cooperate in
the inhibition of cell cycle progression and induction of differentiation in HL-60
leukemic cells. Nutr Cancer 1999;33:105-112.
7. Ito Y, Suzuki K, Suzuki S, et al. Serum antioxidants and subsequent mortality rates of
all causes or cancer among rural Japanese inhabitants. Int J Vitam Nutr
Res 2002;72:237-250.
8. Sharoni Y, Giron E, Rise M, Levy J. Effects of lycopene-enriched tomato oleoresin
on 7,12-dimethyl-benz[a]anthracene-induced rat mammary tumors. Cancer Detect
Prev 1997;21:118-123.
9. Pool-Zobel BL, Bub A, Muller H, et al. Consumption of vegetables reduces genetic
damage in humans: first results of a human intervention trial with carotenoid-rich
foods. Carcinogenesis 1997;18:1847-50.
10. Bohm F, Tinkeler JH, Truscott TG. Carotenoids protect against cell membrane
damage by the nitrogendioxide radical. Nat Med 1995;1:98-99.
11. Agarwal S, Rao AV. Tomato lycopene and low density lipoprotein oxidation. A
human dietary intervention study. Lipids 1998;33:981-4.
12. Arab L, Steck S. Lycopene and cardiovascular disease. Am J Clin Nutr 2000;
71(suppl),169:15-55.
13. Arab, Lenore, Susan S. Lycopene and cardiovascular disease. Ame J clin nutr
2000 (suppl)19613-53.
14. Bohm Y, Bitsch R. Intestinal absorption of lycopene from different matrices
and interactions to other cariotenoids, the lupid status and the antioxidant
capacity of human plasma. Eur J Nutr 1999;38:118-25.
15. Boileau AC, Merchen NR,Wasson LE, et al. Cis lycopene is more bioavilable than
Trans-lycopene in vitro and in vivo lymph cannulated ferrets. J Nutr
1999;129:17681.
16. Clinton SK, Emenhiser C, Schwartzm SJ, et al. cis trans lycopene isomers,
corotenoids and retinol in the human prostate. Cancer Epidemiol Biomarkers
Prev 1996;5:823-33.
17. Clinton SK. Lycopene chemistry, biology and implecation for human health and
disease. Nutr Rev 1998;56:35-51.
18. Heber D, Lu Qy. Overview of mechanism of action of lycopene. Exp biol med
(Maywood) 2002;227:920-3.
19. Agrawal RC, Kumar S. Preventive of cyclophophamide induced micronuclear
formation in mouce bone marrow by Indole -3-carbinol. Food and chemical toxical
1998;36:975-7.
20. Bohm F, Tinkeler JH, Truscott TG. Carotenoids protect against cell membrane
damage by the nitrogendioxide radical. Nat Med 1995;1:98-99.
21. Karim I, Marilene P,Cory B, Anil KJ. Deficency of NRH:Quinine Oxidoreductase
2 Increase susceptibility to 7,12-Dimethyl benz(a)anthracene and Benzo(a)Pyreneinduced skin carcinogenesis. Cancer Research 2004;64:5925-8.