DIETARY PHYTATE-IRON INTERACTION : ROLE ON

SUPPRESSING COLON CANCER

Presented By
Satya Prasad PPV
Ph.D. Research Scholar
Department Of Biotechnology
Krishna University
Machilipatnam-521001
Email: satyabiotech.prasad@gmail.com

Name, Designation And Address Of Guide:

Dr B. Vijaya Lakshmi,
Assistant Professor,
Institute Of Genetics and Hospitals,
Osmania University, Ameerpet,
Hyderabad
Phone: 9293720419
Email: bodigavijayasri@gmail.com
 INTRODUCTION TO COLON CANCER
 Colon cancer is third most common type of cancer and second most frequent
cause of cancer-related death

 The etiology and molecular pathogenesis of colorectal cancer is complex and

multi factorial

 Dietary nutrients playing a key role in the development and progression of

colorectal cancer

 A disease in which normal cells in the lining of the colon or rectum begin to
change, grow without control, and no longer die. Usually begins as a
noncancerous polyp that can, over time, become a cancerous tumor.

 The Symptoms of Colorectal Cancer A change in bowel habits: diarrhea,

constipation, or a feeling that the bowel does not empty completely. Bright red or
dark blood in the stool Stools that appear narrower or thinner than usual
Discomfort in the abdomen, including frequent gas pains, bloating, fullness, and
cramps Unexplained weight loss, constant tiredness, or unexplained anemia (iron
deficiency).
Cancer Treatment: Surgery, Chemotherapy, Radiation, Therapy and Targeted
Therapy
 COLORECTAL CANCER STAGING

Stage 0 Colorectal Cancer

Stage I Colorectal Cancer

Stage II Colorectal Cancer

Stage III Colorectal Cancer

Stage IV Colorectal Cancer

Survival Rate:
stage I 74%, stage II 67%, stage III 46%, stage IV 6%
Risk Factors Of Colorectal Cancer
 EFFECT OF PHYTATE

 In mammals, Phytate and its lower phosphorylated derivates participate

in regulating vital cellular functions, particularly cell division and
differentiation.
 Phytate has also been reported to possess antioxidant, anti-
inflammatory and immune enhancing function. The most striking effect
of PA has been documented in cancer prevention by controlling tumor
growth, progression and metastasis
 Phytate widely occurs in plant seeds, and grains, tubers, and
vegetables.
 Large fraction of unabsorbed trace elements entre into the colon and
promote cancer progression.
 As the phytate content in the diet increase, absorption of intestinal iron
come down.
 PHYTATE STRUCTURE AND ABSORPTION OF
Fe, Zn, Ca, Mg
Phytate has high density of negatively charged phosphate
group and it forms stable complexes with positively charged
proteins, amino acids and minerals in food.
PROPERTIES OF PHYTIC ACID
 INFLUENCE OF IRON ON CARCINOGENESIS

 Iron is an essential micronutrient required for various cellular

processes.
 A recent meta-analysis also supports a positive dose-response
correlation of dietary iron levels with CRC risk
 The relationship between iron and colon cancer is more obvious due
to the fractional iron absorption and high levels of unabsorbed
luminal iron interacting with the colon epithelium.
 Iron is an essential micronutrient required for various cellular
processes, including erythropoiesis, cell proliferation.
 Laboratory experimental studies suggest iron and may increase risk
of colon cancer.
 There is a positive correlation between iron and colorectal cancer
risk.
 These dietary iron and iron stores essential nutrients for colon
cancer.
 In epidemiological laboratory studies result of high levels of iron
results leads to colon cancer tissues.
 HYPOTHESIS

“ Dietary Phytate Suppresses AOM Induced

Colon Cancer by Lowering Iron Bioavailability
in the Colon Epithelium”.

 This will be tested in the animals that are receiving 1% and

2% phytic acid supplementation after tumor induction by
analyzing the following parameters
 OBJECTIVES
 To study the potential changes in the levels of iron in aberrant crypt foci
(pre-neoplastic lesions) and their body Fe stores at 20 weeks

 To study the sustained changes in the levels of iron and during

progression from aberrant crypt foci into colonic tumors and total body Fe
stores at 36 weeks.

 To characterize the respective changes in transporters and storage

protein expression induced by azoxymethane

 Elucidate the probable mechanisms of changes in the redox status of

normal colon as opposed to aberrant crypts and colonic tumors

 Assess the reversibility/preventability of colonic tumor formation by dietary

phytate supplementation and to understand the underlying mechanisms
 STUDY DESIGN
A total of 48 five-week-old male Fisher 344 rats taken.

Hygienic conditions were maintained by weekly changes of woodchip beds.

The rats were randomly assigned to control (n = 32) and AOM (n=32) groups.

After 20 weeks, few animals were sacrificed and observe for aberrant crypt
foci formation in the colon. Samples have been collected and
histological/biochemical analysis is in progress.

The remaining animals (n =24) in AOM group were randomly divided into 3
groups and fed either 0, 1 or 2% phytate containing diet for the next 16 weeks
(n =8, each).

The effect of dietary phytate on suppression of colonic ACF and their

progression into colonic tumours was studied after 16 weeks of phytate
consumption.
 Animal Experment
Fishers 344 Rats (N=64, 5 Week Old, Male)
16 Rats 32 Rats
Group A (AIN-93) saline Group B (AIN -93) AOM
(15 mg/kg i.p)
8 animals from each group sacrificed after 20 weeks
For ACF formation
Remaining rats with control diet Remaining rats with phytate diet

Control AOM
n=8 for each group n=8 for each group
0% 0% 1% 2%

After 16 weeks all Rats were sacrificed by CO2 asphyxiation.

observed for the effect of phytate on colonic ACF suppression with different
biochemical , molecular and pathological studies.
 DIET PREPARATION
powdered diet prepared according to AIN-93 recommendation
daily food intake and weekly body weights are recorded
Diet components controle diet 1% phytate diet 2% phytate diet

starch 54.5% 53.5% 52.5%

casein 25% 25% 25%
Oil 10% 10% 10%
cellulose 5% 5% 5%
Vitamin mixture 1% 1% 1%

Mineral mixture 4% 4% 4%

L-cystain 0.3% 0.3% 0.3%

Choline chloride 0.2% 0.2% 0.2%

phytate 0% 1% 2%
 CARCINOGEN (AOM) INJECTION
 Azoxymethane (AOM) is a potent carcinogen used to induce colon
cancer in rats and mice. It has been used in studies evaluating
efficacy of preventative treatment for azoxymethane-induced
carcinogenesis.

 Azoxymethane is also commonly used to determine the

chemopreventative effectiveness of particular foods such as
undigestable sugars, red meat, and green tea among others in
rodent models. These rodent model results aid in the identification
of possible preventative approaches to human colon cancer.

 The cyclooxygenase 2 (COX-2) inhibitor NS-398 (Product

No. reduces the incidence of preneoplastic cells in rats treated with
azoxymethane.
 SAMPLE COLLECTION

Aliquots of blood were obtained with EDTA as anticoagulant to measure

the hematological parameters.

blood was centrifuged at 1,500 × g for 15 min at 4°C without

anticoagulant to separate the red blood cells (RBC) from the serum

ABERRANT CRYPT FOCI FORMATION:

The colons were longitudinally opened, rinsed with saline and fixed in 10%
buffered formalin for at least 24 h. The colons were stained with 0.2%
methylene blue for 10 min.

HISTOCHEMICAL ANALYSIS: For tumor assessment at the end of 36

weeks, colon tissue was removed, dissected longitudinally, flushed with
phosphate-buffered saline (PBS), fixed in 10% (v/v) neutral buffered
formalin prior to staining with hematoxylin and eosin (H&E).
 Parameters to be analyzed
Fe Determination in the Diets and Liver
Samples of diets and liver (1 g of DW) were mineralized and Iron
concentrations in the different diets were determined by atomic absorption
spectrophotometry

Hematological tests:
Hemoglobin and hematocrit concentrations were measured using an
automated hematology analyzer

Serum Ferritin:
Serum ferritin concentration was determined using the Rat Ferritin ELISA
Kit

Serum Fe, TIBC, and Transferrin Saturation:

The rate of transferrin saturation, serum Fe concentration and TIBC were
determined using Sigma Diagnostics Iron and TIBC reagents

Serum Hepsidin:
Hepcidin-25 concentration was determined using a ELISA Kit
 Parameters to be analyzed
DNA extraction from paraffin –embedded Tissues
Each 10-m section cut from the paraffin blocks and colonic DNA extracted
was mixed with 300 l of lysis buffer

Restriction endonuclease mediated selective polymerase chain

reaction (REMS-PCR) for mutational analysis
Mutations at the first and second bases of codon 12 of the K-ras gene
were detected using REMS-PCR.

Electron spin resonance spectroscopy

In order to understand the nature and intensity of free radical species
produced in AOM-administered rat colon mucosa
 ACF ANALYSIS AND TUMOUR ASSESSMENT

 Colons were longitudinally opened, rinsed with saline and

fixed in 10% buffered formalin for at least 24 h.

Colons were stained with 0.2% methylene blue solution for

10 min.

For tumor assessment at the end of 36 weeks, colon tissue

was removed, dissected longitudinally, flushed with
phosphate-buffered saline (PBS), fixed in 10% (v/v) neutral
buffered formalin prior to staining with hematoxylin and eosin
(H&E).
 APOPTOSIS-TUNNEL KIT

 In situ nick-end labeling (TUNEL) of fragmented DNA

was performed on paraffin tissue sections.

 The percentage of positive apoptotic cells was calculated

after brown and green nuclei were counted by an
operator who was blinded to the treatments of each
sample.
Cytoplasmic, membrane, and nuclear fractions were isolated
from colonic mucosa scraped from the serosal layers using the
nuclear and cytoplasmic extraction kit .

30 µg of protein separated on 10% sodium dodecyl sulfate

polyacrylamide gels and transferred to polyvinylidene fluoride
(PVDF) membranes.

. The membranes were incubated with primary antibodies

against target proteins overnight at 4°C.

Primary antibody binding was detected using a (HRP)-

conjugated anti-rabbit IgG antibody and was visualized using
an enhanced chemiluminescence (ECL) detection.
 PROTEINS EXPRESSION BY WESTERN BLOTS

TARGET PROTEINS DETECTED BY WESTERN BLOTTING

Tumor proliferative markers

β-catenin : nuclear and cytoplasmic levels
PCNA cytoplasm

Inflamatory Markers:
Cox-2: cytoplasm

House keeping proteins (internal standards)

GAPDH: cytoplasm
Lamin: nucleus
 IRON TRANSPORTERS

Import proteins:
Divalent Metal Ion Transporter1 (DMT1) , Duodenal
cytochrome B reductase Dcytb), Transferin Receptor
(TfR)

Export proteins
Ferroportin, Hephaestin reductase

Storage proteins
Transferin and Ferritin
 IRON TRANSPORTER PROTEINS EXPRESSION BY
REAL TIME-PCR
 Total RNA was isolated from rat colon mucosa tissues using Rnazol

 An aliquot (1 µg) of RNA was reverse-transcribed with reverse

transcriptase and oligo-dT primers. The forward and reverse primers
for selected genes were designed using Primer Express 2.0.

 Real time PCR was performed using glyceraldehydes-3-phosphate

dehydrogenase RNA as an internal standard, using the probes and
primers listed below.

 The Ct values of interested genes were first normalized with that of

GAPDH in the same sample; the relative differences between
control and treatment groups were then calculated and expressed
as relative increases by setting the control as 100%.
 PRIMER SEQUENCES

Primer Forward 5'————————–3' Reverse 5'————————–3'

Primer sequences used for real-time PCR analysis

Fpn1 TTC CGC ACT TTT CGA GAT GG TAC AGT CGA AGC CCA GGA CTG T

DMT1 GCT GAG CGA AGA TAC CAG CG TGT GCA ACG GCA CAT ACT TG
Dcytb TCC TGA GAG CGA TTG TGT TG TTA ATG GGG CAT AGC CAG AG

TfR1 ATA CGT TCC CCG TTG TTG AGG GGC GGA AAC TGA GTA TGG TTG
A
Hephaestin CAC ATT TTT CCA GCC ACC TT TGA CGA ACT TTG CCT GTG AG

Dcytb TCC TGA GAG CGA TTG TGT TG TTA ATG GGG CAT AGC CAG AG

Transferrin GGC ATC AGA CTC CAG CAT CA GCA GGC CCA TAG GGA TGT T
 8-OHdG adducts in the colon mucosa from AOM-
administered and phytate-supplemented rats

Treatment 8-OHdG/105 dG

Control 0.15±0.07

AOM 0.42*±0.05

AOM+1% Phytate 0.26**±0.03

AOM+ 2% Phytate 0.18**±0.03

Electron spin resonance spectra of DMPO-OH adduct observed in colon mucosa from
AOM-administered rats, with and without different doses of phytate.
All the spectra were recorded using 153 mM DMPO and a receiver gain of 2.0 x 105.
 ACF ANALYSIS AND TUMOUR ASSESSMENT
Normal crypts with Mucin Mucin depleted foci

Normal crypts Aberrant crypts

 ACF ANALYSIS AND TUMOUR ASSESSMENT

Adenoma Adenoma with Atypical Hyperplasia

Adenocarcinoma Invasive adenocarcinoma

 ACF TABLE
Inhibitory effect of phytate on AOM-induced aberrant crypt foci incidence and
multiplicity in Fisher 344 male rat colon
Grou Treatm Inciden No. Crypt multiplicity of Tumor incidence
ps ent ce of ACF/col ACF
ACF on
formati 1 2 3 ≥4 Non- Invasiv
on (%) cry crypt crypt crypt invasiv e (%)
pt s s s e (%)
1 Control 0/8 (0) — — — — — — —

2 8/8 155± 14 52± 65±8 22±6 16±6 5 (62.5) 3(37.5)

AOM (100) 10

3 AOM + 8/8 87 ± 9* 33 ± 26 ± 16 ± 12 ± 2 (25) —

1% (100) 3* 2* 2* 2
Phytate

4 AOM + 8/8 59 ± 8* 21 ± 18 ± 12 ± 8±2

2% (100) 2* 3* 3* — —
Phytate

NOTE: Data are shown as mean ± SD of eight samples in each group.

*P < 0.001, vs AOM (Bonferroni T test).
 APOPTOSIS-TUNNEL KIT

Control AOM

1% PHYTATE 2% PHYTATE
 APOPTOSIS-TUNNEL ASSAY
Apoptotic index in colonic mucosa of Phytate-treated
AOM-induced colon cancer

Treatment Apoptotic cells (%)

Control 3.28 ± 1.04

AOM 2.58 ± 1.12

AOM + 1% phytate 22.50* ± 5.60

AOM + 2% phytate 36.86 **± 6.64

Each value expressed as mean ± SD of three determinations.
Administration of 1% and 2% phytate significantly increased in the
apoptotic cells compared to the AOM-alone group (P<0.05).
TUMOR PROLIFERATIVE MARKERS
 IRON STATUS AND ESTIMATION

Iron status indices in AOM-administered and phytate-fed rats

Control AOM AOM+1% AOM+2%
Phytate Phytate
Hb (g/dL) 15.60±1.24 14.68±1.36 10.2*±1.80 7.46**±1.20
Hct, % 40.2±0.68 39.4±0.60 30.6*±0.50 24.0**±1.54
Serum iron 200.8±20.3 198.5±17.8 122.4*±34.8 56.8**±8.5
(g/dL)
TIBC (g/dL) 600.0±70.8 602.8±60.4 681.2*±60.2 758.0**±75.5
Liver iron (g/g 38.0±7.16 35±3.48 28.0*±2.20 20.4**±1.80
wet tissue)
Serum ferritin 1480.8±65.0 1468.2±60.8 1120.5*±58.0 850.4**±42.8
(ng/mL)
Serum 18.25±0.50 17.6±0.62 13.4*±0.46 12.2*±0.54
hepcidin
(ng/mL)
Each value expressed as mean ± SD of at least 6 determinations. Value in the same row
with different superscript letter indicates significant difference by Tukey test (P<0.05).
Administration of 1% and 2% phytate significantly increased the apoptotic cells
compared to the AOM-alone group (P<0.05).
IRON Content Of Colon Cancer Tissues
IRON TRANSPORT PROTEINS BY WESTERN BLOTS
DMT1,DCytB,TfR1, Ferropottin, Ferritin, Hephaestine CK-19
IRON TRANSPORT PROTEINS By REAL TIME PCR
DMT1,DCytB,TfR1, Ferropottin, Ferritin, Hephstine CK-19
 K-RAS MUTATIONS

FIgure 1. REMS-PCR analysis of K-ras mutations in colon ACF and tumor samples from AOM-administered and phytate-treated rats

AOM AOM + 1% Phytate AOM + 2% Phytate

Ladder ACF tumor tumor ACF ACF tumor ACF ACF ACF Control
250
200 Template
150
100
Amplicon
50
Automated direct sequencing of codon 12 mutations of K-ras gene.
The amplified PCR products were sequenced to confirm
the base composition of mutated and unmuated DNA at codon 12.
 SUMMARY
SummarySummary:
•Two doses of azoxymethane(AOM)@ 15mg/kg body weight successfully induced
37.5% invasive and 62.5% non-invasive colonic tumorsafter 36 weeks inFisher 344
male rats.
•AOM- induced preneoplastic lesions showed increased expression of β-cateninand
strong nuclear localization and proliferating cell nuclear antigen (PCNA),that are
involved in altered signaling during proliferation . It also enhanced the expression of
inflammatory protein cyclooxygenase (COX-2).
•Dietary phytate supplementation at 1 and 2% during post initiation showed a
significant decrease in the number of aberrant crypt foci (ACF) in a dose dependent
manner.
•Phytate supplementation lowered the expression of tumor proliferative and
inflammatory markers, such as β- catenin, PCNA and COX-2. More so, the nuclear
localization of β-catenin is decreased.
•Azoxymethane as such did not affect the total body iron stores and iron binding
capacity. Whereas, increased accumulation of free and bound iron levels in the
colonic cancer tissue has been observed.
 SUMMARY

•Phytate supplementation significantly depleted body iron status. Serum ferritin and
hepcidin levels decreased with phytate, indicating reduced iron absorption.
•Various proteins that regulate the import, accumulation and export of iron have been
altered during carcinogenesis.
•Expression of iron import proteins i.e. DMT1, DCytb, TfR has been upregulated and
export proteins i.e. Hephaestinand ferroportin expression has been downregulated
during tumor formation, suggesting increased iron requirements. This lead to
accumulation of iron in the form of ferritin in the enterocytes.
•Phytate supplementation lowered the expression of DMT1,DCytb,TfR expression
and upregulatedhephaestin and ferroprotein levels.
•Phytate corrected the imbalance between the importer and export protein
expression.
•Phytate limited the iron bioavailability and suppressed tumor cell proliferation.
•Increased formation of 8-OHdG and k-ras mutation frequency has been observed in
AOM-induced tumors.
•As, Iron is a pro-oxidant and increases free radical formation during tumor
progression. Phytate a well established iron-chelator and antioxidant, lowered 8-
0HdG levels and k-ras mutation frequency in colonic epithelial cells.
 Paper 1
Dietary phytate reduces K-ras mutational frequency and hydroxyl radical formation in
azoxymethane-induced colon cancer

Poorna Venkata Satya Prasad Pallem1, Sreedhar Bodiga2, Vijaya Lakshmi Bodiga3*
1Department of Biotechnology, Krishna University, Machilipatnam.
2Department of Biochemistry, Kakatiya University, Warangal.
3Institute of Genetics & Hospital for Genetic Diseases, Begumpet, Osmania University, Hyderabad.

Biochemical and biophysics research comuncaions (BBRC)

Manuscript number BBRC-17-4741

Paper 2
Dietary phytate suppresses/modulates the changes in iron transport protein expression
induced by azoxymethane in colon carcinogenesis

Poorna Venkata Satya Prasad Pallem1, Sreedhar Bodiga2, Vijaya Lakshmi Bodiga3*
1Department of Biotechnology, Krishna University, Machilipatnam.
2Department of Biochemistry, Kakatiya University, Warangal.
3Institute of Genetics & Hospital for Genetic Diseases, Begumpet, Osmania University, Hyderabad.

Journal of applied Pharmaceutical science

Manuscript number JAPS-2017-06-602
 ACKNOWLEDGEMENT
Project Code:
Project # IT PR3391/FNS/20/528/2011 (DBT)

Project Name:
Dietary Phytate-Mineral Interaction: role in Suppressing Colon Cancer

DEPARTMENT OF BIOTECHNOLOGY
KRISHNA UNIVERSITY
MACHILIPATNAM-520001
AND
ENDOCRINOLOGY AND METABOLISM DEPARTMENT
NATIONAL INSTITUTE OF NUTRITION
HYDERABAD
Satya prasad ppv
Dept of biotechnology
Krishna university
Machilipatnam
Histopathology of colonic lesions developed in rats treated with AOM.
 ACF DETECTION

WHOLEMOUNT PROCEDURE

 Aberrant crypt foci (ACF) represent the earliest identifiable intermediate

precancerous lesions during colon carcinogenesis in both laboratory animals and
humans

colon jejunum and duodenum are cut opened and pinned on formalin paraffin
platform and incubate 24 hors in cold room

0.1 % methylene blue was filtered with blotting paper after dilution in M.Q.
10 mints incubate in methylene blue solution

Rinse under tape water and observe under microscope.

After observation all samples are sis rolled and stored in formalin for tissue
processing
A 4X4 A 4X5

A 4X6 A 4X7
 ALCIAN BLUE STAINING
Alcian Blue Solution (pH 2.5):
Alcian blue, 8GX -------------------- 1 g
Acetic acid, 3% solution ----------- 100 ml
Mix well and adjust pH to 2.5 using acetic acid.
0.1% Nuclear Fast Red Solution:
Nuclear fast red ------------------- 0.1 g
Aluminum sulfate------------------ 5 g
Distilled water ---------------------100 ml
Dissolve aluminum sulfate in water.
Add nuclear fast red and slowly heat to boil and cool.
Filter and add a grain of thymol as a preservative.
Procedure:
 Deparaffinize slides and hydrate to distilled water.
 Stain in alcian blue solution for 30 minutes.
 Wash in running tap water for 2 minutes.
 Rinse in distilled water.
 Counter stain in nuclear fast red solution for 5 minutes.
 Wash in running tap water for 1 minute.
 Dehydrate and through 95% alcohol, 2 changes of absolute alcohol, 3 minutes each.
ALCIAN BLUE_002 ALCIAN BLUE_003

ALCIAN BLUE_005 ALCIAN BLUE_008

ALCIAN BLUE_02 ALCIAN BLUE_011

PRESSIAN BLUE_003 PRESSIAN BLUE_005

 PRUSSION BLUE STAING
5% Potassium Ferro cyanide: Potassium ferrocyanide 25.0 gm.Distilled water 500.0 ml
5% Hydrochloric Acid: Hydrochloric acid, conc. 25.0 ml, Distilled water 475.0 ml
Working Solution:
5% potassium ferrocyanide 25.0 ml
5% hydrochloric acid 25.0 ml
Make fresh, discard after use.
PROCEDURE:
1. Deparaffinize and hydrate to distilled water.
2. *Working solution, * microwave, 30 seconds. Allow slides to stand in
solution for 5 minutes, in the fume hood.
3. Rinse in distilled water.
4. Nuclear-fast red, 5 minutes.
5. Wash in tap water.
6. Dehydrate, clear, and cover slip.
*Conventional method: room temperature for 30 minutes.
RESULTS:
Iron (hemosiderin) blue
Nuclei red
Background pink
PRESSIAN BLUE_008 PRESSIAN BLUE_0011

PRESSIAN BLUE_015 PRESSIAN BLUE_016

 IRON ANALYSIS by AAS (Shimadzu AA-7000)
LIVER, SERUM, COLON, DUODENUM, JEJUNUM, IRON,
and AAS ANALYSIS:
AAS SAMPLE PREPARATION:

Take 50 µl of serum and etc into 2ml of eppendorf tube

Add 4 µl of HNO3 concentration 1:1
Heat or boil for 10 mints

Make up with to 0.5 ml with M.Q.

Centrifuge at 3,500 rpm for 10 mints.

Collect supernatant and make measure with AAS

 STANDARDS PREPARATION
IRON SATNADARS FOR FLAME AND GRAPHITE
 Iron standard is dissolved in nitric acid
 Solution concentration is 1mg/ml
 FOR FLAME (mg)
 Take 100 µl of standard in to 9,900 µl =100 µg/ml or 10 µl into 990 µl=100
µg/ml
 Take 100 µg solution as serial
 50 µl in 9,950 M.Q. or 5 µl in 995 M.Q. for 0.5 µg
 100 µl in 9,900 M.Q. or 10 µl in 990 M.Q. for 1 µg
 200 µl in 9,800 M.Q. or 20 µl in 980 M.Q. for 2 µg
 300 µl in 9,700 M.Q. or 30 µl in 970 M.Q. for 3 µg
 400 µl in 9,600 M.Q. or 40 µl in 960 M.Q. for 4 µg

( 1 ml make up sample for micro sampling)

 FOR GRAPHITE (ng)
 Take 100 µl of standard in to 9,900 µl =100 µg/ml or 10 µl into
990 µl=100 µg/ml
 Take 100 µg of standard solution in to 9,900 µl=100 ng/ml
 Or
 10 µl of 100 µg standard solution in 990 µl=100 ng
 50 µl in 9,950 M.Q. or 5 µl in 995 M.Q. for 0.5 ng
 100 µl in 9,900 M.Q. or 10 µl in 990 M.Q. for 1 ng
 200 µl in 9,800 M.Q. or 20 µl in 980 M.Q. for 2 ng
 300 µl in 9,700 M.Q. or 30 µl in 970 M.Q. for 3 ng
 400 µl in 9,600 M.Q. or 40 µl in 960 M.Q. for 4 ng

 Parameters being / to be determined:

1. Cell cycle dynamics during tumor induction

2. Iron and nutritional status in tumor induced rats
3. Biochemical and molecular mechanism involved in altered nutritional
status of trace elements
4. Colonic epithelial tissue redox status
 SUMMARY
 Successful demonstration of aberrant crypt foci formation with two doses of
azoxymethane subcutaneous injections (one dose/week @ 15mg/kg body
weight)
 Iron concentrations were lowered at the site i.e. colonic epithelial cells,
whereas the storage and circulatory levels of iron increased during colon cancer in
F344 rats.
 Iron concentrations are relatively high at the site i.e. colonic epithelial cells,
and also the circulatory levels whereas the liver lowered during colon cancer
initiation in F344 rats.
Role of phytic acid in chemoprevention of colon cancer
 In order to understand the mechanism involved in anti-cancer action of
phytate, aberrant crypt foci number, cell proliferation, differentiation and cell
death will be monitored.
 Iron concentrations in tumor, normal tissues and body stores will also be
monitored to verify whether the observed change in cell cycle is due to altered
mineral nutritional status during phytic acid
 AKNOWLEDGEMENT

1.Reddy BS. Studies with the azoxymethane-rat preclinical model for assessing colon tumor
development and chemoprevention. Environ Mol Mutagen 2004; 44: 26-35
2. Nelson, RL. Dietary iron and colorectal cancer risk. Free Radical Biol. Med., 1992; 12:
161–168.
3. Knekt P, Reunanen A, Takkunen H, Aromaa A, Heliovaara M, Hakulinen T. Body iron
stores and risk of cancer. Int. J. Cancer 1994; 56: 379–382.
4. Stevens RG, Jones DY, Micozzi MS, Taylor PR. Body iron stores and the risk of cancer. N.
Engl. J. Med., 1988; 319: 1047–1052.
14
5. Stevens RG, Graubard BI, Micozzi MS, Neriishi K, Blumberg BS. Moderate elevation of
body iron level and increased risk of cancer occurrence and death. Int. J. Cancer. 1994; 56:
364–369.
6. Cherian MG, Huang PC, Klaasen CD, Liu YP, Longfellow DG, Waalkes, MP. National Cancer
Institute workshop on the possible roles of metallothionein in carcinogenesis. Cancer Res.
1993; 53: 922-925.
7. Shamsuddin AM, Ullah A. Inositol hexaphosphate inhibits large intestinal cancer in F344
rats 5 months after induction by azoxymethane. Carcinogenesis 1989; 10: 625-626.
8. Balasubramanian, KA, Manohar, M and Mathan, VI (1988). An unidentified inhibitor of
lipid peroxidation in intestinal mucosa. Biochim Biophysic Acta, 962: 51-58.
 METHODS AND METHODOLOGY
Animals and housing:
Sixty four number of two month-old male Fisher 344 rats were obtained from National
Centre for Laboratory Animal Sciences (NCLAS).

The animals were housed individually in a thermally controlled environment (22°C ± 2)

with 12: 12 light dark cycle and given free access to water and food as described

These conditions, as well as treatment of the animals met the guidelines set by the
CPCSEA and all protocols were approved by Institutional Animal Ethics Committee (IAEC).

A record of daily food intake and weekly body weights of all the groups was kept
throughout the study period.

SERUM SEPARATION:
Blood drawing is the first step to killing rats before co2 incubator.
Taken all blood sample in 15 ml falcon tube rotor
12000 rpm in 10mints.
Precipitated serum is separated into 0.5ml of eppendorf tubes.