EFFECT OF FREQUENCY OF SMOKING

EXPOSURE AND COMBINED TREATMENT

VITAMIN C AND BETA CAROTENE ON
HISTOPATHOLOGICAL CHANGES AND P53
EXPRESSION IN RAT BLADDER (Rattus
Norvegicus)
By :
Rudy Eka Arethusa Putra
Supervisor:
dr. Taufiq Nur Budaya, Sp.U
Dr. dr. Besut Daryanto, Sp.B, Sp.U (K)
CHAPTER 1
BACKGROUND
• RSCM and RSKD Jakarta  340 bladder cancer patient (1995-2004), increase 15%/ year
(mean age 54 y.o) (Abdih et al, 2014)
• RSSA Malang  240 bladder cancer patient (January 2012-Desember 2018)
• Smoker has higher risk compared to non-smokers (Freedman et al, 2011)
• P53 deficiency  resistant to chemotherapy (Aubrey et al., 2016)
• Intravenous nicotine administration  decrease p53 expression significantly at lung,
hepar, and bladder (Asiri, 2010)
• Vitamin C decrease nicotine-induced elevation of p53 expression in rats (Ahmed et al.,
2014)
• Low dose beta carotene decrease p53 expression in cigarette-induced rats & has
protective effect on malignancy changes in lung tissue (Liu et al., 2004)
PROBLEM FORMULATION
Does the increased frequency of smoking exposure affect the
histopathological features (dysplasia and chronic inflammation score) and
reduce p53 expression in bladder mucosa of Wistar rats?

Does the discontinuation of smoking exposure affect the histopathological

features (dysplasia and chronic inflammation score) and p53 expression in
bladder mucosa of Wistar rats?

Does the combined treatment vitamin C and beta carotene affect the
histopathological features (dysplasia and chronic inflammation score) and
improve p53 expression in bladder mucosa of smoking-induced Wistar rats?
AIM OF STUDY
• To investigate the effect of increased frequency of smoking exposure
affect the histopathological features (dysplasia and chronic
inflammation score) and reduce p53 expression in bladder mucosa of
Wistar rats?
• To investigate the effect of discontinuation of smoking exposure on
the histopathological features (dysplasia and chronic inflammation
score) and p53 expression in bladder mucosa of Wistar rats?
• To investigate the effect of combined treatment vitamin C and beta
carotene on the histopathological features (dysplasia and chronic
inflammation score) and improve p53 expression in bladder mucosa
of smoking-induced Wistar rats?
CHAPTER 2
BLADDER CANCER
• The American Cancer Society  incidence of bladder cancer 81,190, mortality
rate 17,240 (Fujiyama et al, 2007)
• Incidence in Indonesia is estimated as 5,8 per 100.000 population
(GLOBOCAN, 2008). Data from RSHS Bandung (within last 7 years): 351 cases,
253 cases (72%) is transitional cell carcinoma (TCC). Mean of age 60,8 y.o,
men: women ratio 6:1 (Safriadi et al, 2014)
• The highest risk factor for bladder cancer is smoking, causing 50-65 % cases in
men and 20-30% cases in women (Witjes et al, 2013; Freedman et al, 2011)
• KLASIFIKASI HISTOPATOLOGIK KANKER BULI BULI
T Tumor Primer
Tx Tumor primer tidak dapat dinilai Grading WHO tahun 1973
To Tidak ditemukan tumor primer
Ta Karsinoma papilari non-invasif Papiloma urotelial / (urothelial papiloma)
Tis Karsinoma in situ: “tumor sel datar” Grade 1: differensiasi baik / (well differentiated)
T1 Tumor menginvasi jaringan ikat subepitel
T2 Tumor mengivasi otot Grade 2: differensiasi sedang / (moderately differentiated)
T2a Tumor menginvasi otot superfisial (1/2 luar) Grade 3: differensiasi buruk / (poorly difefrentiated)
T2b Tumor menginvasi otot dalam (1/2 dalam)
T3 Tumor menginvasi jaringan perivesika
T3a Secara mikroskopis
T3b Secara makroskopis (massa ekstravesika) Grading WHO tahun 2004
T4 Tumor menginvasi salah satu dari: prostat, uterus, vagina, dinding pelvis, dinding
abdomen Lesi datar / Flat lesions
T4a Tumor menginvasi prostat, uterus, atau vagina Hiperplasia (lesi datar tanpa atipia atau aspek papilari)
T4b Tumor menginvasi dinding pelvik atau dinding abdomen Atipia reaktif (lesi datar dengan atipia)
Atipia dengan signifikansi yang tidak jelas
Displasia urotelial
CIS urotelial
Lesi papiler
N Kelejar Limfe
Nx Kelenjar linfa regional tidak dapat dinilai Papiloma urotelial (lesi jinak total)
No Tidak ditemukan metastasis kelenjar limfe regional Neoplasma urotelial papilari dengan potensi keganasan rendah / Papillary
N1 Metastasis ke satu kelenjar limfe regional di true pelvis (hipogastrik, obturator,
iliaka eksternal, atau presakral
urothelial neoplasm of low malignant potential (PUNLMP)
N2 Metastasis ke kelenjar limfe regional multipel (hipogastrik, obturator, iliaka Karsinoma urotelial papiler grade rendah
eksternal, atau presakral Karsinoma urotelial papiler grade tinggi
N3 Metastasis ke kelenjar limfe di iliaka komunis
M Metastasis
Mx Metastasis tidak dapat dinilai
Mo Tidak ada metastatis
M1 Ditemukan metastasis
P53
• Structure and Function
• Protein p53 (p53)  tumor-suppressor encoded by p53 gene, located in short arm
of chromosome 17, has 393 amino acid and 5 domain 13 (Benjamin et al, 2007).
• p53  transcription factor regulating cell cycle and often mutated in almost
malignancies, repair DNA damage, DNA synthesis, cell differentiation, modulates
cell apoptosis (Leffel et al, 2000; Prakoeswa, 2008)

Struktur p53 (Bai and Zhu, 2006)

Fungsi p53 (Bai and Zhu, 2006)

SMOKING EXPOSURE AND HISTOPATHOLOGICAL
CHANGES RELATED TO CANCER

Hubungan Rokok Tembakau dengan Kanker (Office of the Surgeon General, 2004)
SMOKING EXPOSURE AND P53
EXPRESSION

Tembakau mensupresi protein proapoptosis dan tumor-suppressor protein (CDC, 2010)

Deteksi imunohistokimia menunjukkan
reaktivitas p53 pada karsinoma sel transisional
(a) Invasive transitional cell carcinoma, poorly
differentiated (Grade III)  tidak
terdeteksinya nuclear p53 (Score (−), (10X))
(b) Papillary transitional cell carcinoma, well
differentiated (Grade I)  sedikit
imunoreaktivitas p53 pada nucleus tumor
(Score (+/−), tanda panah, (10X dan 40X))
(c) Invasive transitional cell carcinoma, poorly
differentiated (Grade II)  imonoreaktivitas
heterogen p53 nuclear (Score (+); arrowed,
(40X))
(d) Papillary transitional cell carcinoma, well
differentiated (Grade I)  imunoreaktivitas
p53 nuclear homogen densitas tinggi (Score
(++); panah kuning, (10X dan 40X), (Panah
merah  jaringan stroma sekitar dan infiltrasi
limfosit tanpa imunoreaktivitas p53 nuclear
(Al-Kashwan et al, 2012)
VITAMIN C
• Vitamin C has 2 isomers: ascorbic acid (vitamin C) (reduced state) and dehydroascorbic acid (DHA)
(oxidized state)  tissue protection against oxidative stress (Jelodar et al., 2013).
• Oxidative stress  imbalance of ROS production and both enzymatic antioxidant defence
mechanism (superoxide dismutase, catalase, dan glutathione peroxidase) and non enzymatic
(vitamin A, C and E) (Burton and Jauniaux, 2011).
• Ascorbate directly scavenge ROS and RNS during normal cellular metabolism (Lee et al., 2004).
• High intake of vitamin C is associated with decreased risk of various cancer such as pharynx, oral
cavity, esophagus, lung, and gastric (Jacob and Sotoudeh, 2002).
• Vitamin C has protective effect on molecular destruction related to carcinogenesis  vitamin C
modulates signal transduction and gene expression (Li and Schellhorn, 2007).
BETA CAROTENE
• ß-Carotene  secondary metabolite synthesized by various plants
• ß-Carotene  unoxidized compounds of carotenoid, its structure is
identical with retinol and shows highest bioactivity as the precursor of
vitamin A (Fratianni et al., 2010)
• Low dose beta carotene decrease p53 expression in smoking-induced
rats and protect lung tissue from malignancy changes (Liu et al., 2004)
CHAPTE
R3
 Wistar rats Smoking

nAChRs Nicotine

Smoking Vitamin C
discontinuation Beta Carotene

ROS ↑ Bcl-2 ↓

Investigated
JNK Pathway DNA Damage Bax ↑
Not investigated
AP-1 P53 expression ↓ Bax/Bcl-2 ratio ↑ Cause

Bind
CTGF Apoptosis ↑

Proliferation of urothelial or transitional

cell Altered bladder histology
HYPOTHESIS
• Increased frequency of smoking exposure induce dysplasia and
increase chronic inflammation score and reuce of p53 expression in
bladder mucosa of Wistar rats.
• Discontinuation of smoking exposure reduce dysplasia and chronic
inflammation score and increase p53 expression in bladder mucosa of
Wistar rats.
• Combined treatment of vitamin C and beta carotene inhibit dysplasia
and reduce chronic inflammation score and improve p53 expression in
bladder mucosa of smoking-induced Wistar rats.
CHAPTE
R4
DESIGN
True experimental post-test-only controlled group design

TIME AND LOCATION

Pharmacology Laboratory, Pathology Anatomy Laboratory, Biomedical Laboratory,
Faculty of Medicine Universitas Brawijaya (May-September 2019).
POPULATION AND SAMPLE
• 35 Wistar rats divided into 7 groups
• Inclusion criteria:
• Adult healthy Wistar rats
• Male, aged 6-8 weeks
• Body weight 200-250 grams
• Exclusion criteria:
• Not actively moved
• Any anatomical disabled or frizzy hair
VARIABLES
• Independent variables
• Frequency of smoking exposure (0, 1, 2, 4, and 8 times), smoking
discontinuation, treatment with vitamin C and beta carotene
• Dependent variables
• Chronic inflammation score, p53 expression
DEFINISI OPERASIONAL
• Tikus Wistar: Wistar rat Rattus norvegicus species from Lab. Farmakologi FKUB
• Smoking pump: Specified tool for smoke exposure from Pharmacology Laboratory of Brawijaya
University.
• Hematoxylin Eosin Staining: A staining process to evaluate histopathological tissue conduncted
at Pathologi-Anatomy Laboratory of Brawijaya University.
• ImageJ: a free downloaded software to help assess number of cell and also to evaluate their
expression.
• Imunohistokimia: a procedure conducted to assess specified protein by using a specified
monoclonal antibody against p53 (in this study) and their immunostaining kit.
• Smoke: this study using smoke from Surya brand cigarretes.
• Vitamin C and beta carotene: vitamin C and beta carotene provided in tablets which are
dissolved with water and afterward administered via intragastric.
 Variables Operational Definition Tools Variable Scale Unit
Smoke Exposure Smoke exposure is conducted by Frequancy of Numeric Times
smoking pump device in 15 minutes exposure
doses with varied interval daily start
from once, twice, four times and
eight times daily.
Vitamin C and Beta Vitamin C 100 mg/kgBB/day (Milica et Dose and duration Nominal mg
Carotene al., 2016) and beta carotene 430 of administration
Administration mg/kgBB/day (Liu et al., 2004)

Histopathology Chronic inflammation score (Cavali et HE staining, Numeric and Inflammation

al., 2014) and dysplasia evaluated by using Nominal score, positive or
light microscope negative (dysplasia)
P53 Expression Number of cells expressing p53, Stained by Numeric Number of cells
indicated by brown color on immunohistochemi
immunohistochemistry specimen stry method,
evaluated by light
microscope and
software ImageJ
 TREATMENT GROUP
DAYS
GRO 1 2 3 4 5 6 7 8 9 … 60 61 61- 91
90
UP A            
B            
C            
D            
E            
F             
G © © © © © © © © © © © 
: 8x/day : 4x/day : 2x/day : 1x/day : no : sacrificed ©: vit C + β
smoke carotene
Measurement of chronic inflammation score in bladder (Cavalli et al, 2014)
RESEARCH FLOW CHART
Acclimatization (7-14 days)

Randomization

Smoke Smoke Smoke Smoke Without Smoke

Smoke
8x/day 4x/day 2x/day 1x/day smoke 8x/day
8x/day
60 days 60 days 60 days 60 days 60 days 60 days
+ vit C
 no
& BC 60
smoke
days
30 days

Sacrificed

Fixation and Specimen Preparation

HE staining Immunohistochemistry staining

Chronic inflammation score P53 expression

Statistical analysis
Parameters Previous study
Cigarette smoke exposure Cigarette smoke exposure leads to
quantitative increases in DNA binding
activities of FoxF2 and p53 in lung
tissue (Tharappel et al., 2010)
CS exposure induce p53 mutation and
lead to lung cancer (end point at 7.5
and 9 months) (De Flora et al., 2003)
CS treatment for 60 days induce
histological changes (hyperplasia,
dysplasia) of tongue, pharynx, and
larynx mucosa of rats; no alteration of
p53 expression, increased ki-67
expression (marker for proliferation)
(Semenzati et al., 2012)
Recent study
We investigate the effect of CS on
p53 expression in bladder tissue
We investigate the effect of CS on
p53 expression in bladder tissue,
2 months treatment with CS
We investigate the effect of CS on
p53 expression in bladder tissue,
2 months treatment with CS
Parameters Previous study
Beta carotene treatment High dose beta carotene induce tar-
induced oxidative stress in lung tissue
(Palozza et al., 2006)
Cohort study: beta carotene intake
was inversely associated with risk of
tobacco-related cancer among non
smokers, whereas high beta carotene
intake was directly associated with
risk among smokers (Touvier et al.,
2005)
Dual effect of beta carotene on
smoke-induced lung phosphorylation
of JNK, p38, MAPK, and p53 (Liu et al.,
2004)
Recent study
We use low dose of beta
carotene
Design of this study is true
experimental, use low dose of
beta carotene
We investigate the effect of CS
on p53 expression in bladder
tissue, use low dose beta
carotene
Parameters Previous study
Vitamin C treatment Vitamin C and quercetin treatment alleviates
nicotine-induced (intravenous injection, 3
days) oxidative stress in Wistar rats (blood)
(Paunovic et al., 2016),
Vitamin C is better than selenium in
prevention against nicotine-induced (iv
injection, 7 weeks) oxidative stress including
p53 expression in lung, kidney and liver of
pregnant rats (Hassan et al., 2016)
Vit C supplementation more effective than
selenium on reducing nicotine-induced (sc
injection, 8 weeks) oxidative stress, p53 and
Bcl-2 expression (Ahmed et al., 2014)
Recent study
We use cigarette smoke exposure,
not nicotine injection, vitamin C is
combined with beta carotene
Bladder tissue
We use cigarette smoke exposure,
not nicotine injection, vitamin C is
combined with beta carotene
Bladder tissue
We use cigarette smoke exposure,
not nicotine injection, vitamin C is
combined with beta carotene
Bladder tissue