Documente Academic
Documente Profesional
Documente Cultură
Polyphenols Crude fibres Carbohydrates Fats Protein Saponins Gums Carotene Mineral Alkaloids
Matter
Tannin Flavonoids
Adapted from Ranadive et al (1976) and Raghavan and Baruah (1958) (All components
(Percentage based on dry weight (except moisture)) Jayalakshmi & Mathew (1982)
Catechu:
Catechu or Katha, an additive used mainly in Indian quids is derived from the heartwood
of Acacia catechu. It mainly contains catechu-tannic acid (22-50%) with catechins (13-
33%) and quercetin in small quantities (Felter and Lloyd 1898). Ethanolic extracts of
catechu and pure catechin work as antimutagens and retard the process of nitrosation at
acidic pH by scavenging available nitrite (Nagabhushan et al 1988).
Lime (CaCO3):
Lime used can be shell lime or stone lime. Its addition to the mixture makes it alkaline
upon dissolution in water.
Commercial products also contain various flavouring agents; details of which are not
mentioned on the packages.
So essentially, Pan masala contains most of the ingredients present in a traditional betel
quid (please refer to Table 4) but packaged commercially which has led to increased
consumption of Pan masala in recent years.
1.2.2. Pan masala and oral cancer- Epidemiological data:
Epidemiological studies have linked this increase in Pan masala consumption with
increased incidence of OSF (Oral Submucous Fibrosis)- a potentially debilitating and
precancerous condition in which fibrous patches form on the inner surface of cheeks and
ability to open the mouth progressively decreases (Table 2: early and late signs of OSF).
Earlier it had been regarded more as a disease of the elderly but it is now appearing with
increasing frequency amongst the teenagers due to increased consumption of Pan
masala in this age group. (Gupta and Ray 2004).
Table 2 Clinical features of OSF
Early disease Late signs
Blanching of mucosa Fibrous bands
Intolerance to spicy food Trismus
Petechiae Flattening of palate
Depapillation of tongue Hockey-stick uvula
Oral ulceration Reduced tongue mobility
Leathery mucosa Xerostomia
Taste disturbance Keratosis
Apart from epidemiological data from India, studies from other parts of the world
especially South East Asia also suggest the link between areca nut containing chewing
mixtures and prevalence of OSF and/or cancer (Table 3).
Table 3 Case reports of areca nut chewing causing OSF in various parts of the world.
The problem is also rearing its head in western countries via immigrant populations
(Table 3). Table 4 gives compositions of chewing mixtures or quids practised in different
regions.
Table 4 Compositions of chewing mixtures or quids practised in different regions of the
world. (adapted from IARC Monographs, Vol 85)
Sr Chewing Areca Nut Betel Catechu Slaked
No substance lime
Leaf Inflorescence Stem
1 Areca
2 Betel quid
without
tobacco
3 Pan masala
4 Lao-hwa
(Taiwan)
5 Betel quid
(Taiwan)
6 Stem quid
(Taiwan)
Pan masala by itself has been shown to possess mutagenic and carcinogenic properties
to some extent although chemical entities in Pan masala responsible for these effects
have not yet been identified. So first we will take a look at the evidence for various
biological properties of Pan masala.
Fig. 1
A
N
T P
I R
O O
X O
I X
D I
A D
N A
T N
T
ROS play an important role in carcinogenesis by, causing mutations in cell cycle
Oxidative
regulating genes and affecting cell signalling pathways, ultimately damage
pushing the cell
towards uncontrolled cell division.
Levels of various biomolecules including oxidized biomolecules, antioxidants and
antioxidant enzyme activities are used as molecular indicator/s i.e. biomarkers of
oxidative stress/damage that can be used to measure the extent of oxidative
stress/damage or progress of the disease or the effects of treatment.
1.4. Oxidative stress and its biomarkers:
Oxidative stress biomarkers have long been used as exposure biomarkers for a variety
of substances e.g. arsenic, particulate matter (Bräuner et al 2007), chromium (Wong et
al 2005), etc. These prooxidants cause damage to cellular lipids, proteins and DNA by
oxidising reactions. Such oxidised reaction products can be quantitated and are used as
biomarkers.
Among the most commonly used oxidative stress biomarkers are DNA adducts,
especially 8-hydroxy-2′-deoxyguanosine (8-OHdG) and lipid peroxidation by TBARS
assay. Adduction studies (Table 7), particularly, measure biologically relevant dose of the
substance under investigation (Fig 2). Because they quantitate a specific effect of the
exposure substance they may also open a window in disease causation.
EXPOSURE BIOMARKERS
USED
Markers of exposure Markers of disease
Internal dose of
carcinogenic agent
Fig.2
DNA adducts
Biologically effective
dose Protein adducts
Allelic loss
or gain
Early biological
lesion Chromosomal
aberration
Gene mutation
Lipid peroxidation
Oxidative stress levels vary due to various factors such as diet, exercise, air pollution,
infections, etc. (Halliwell 1999) and according to the metabolism, exposure level of the
specific tissue (Frei et al 1998, Halliwell et al 2000). All of these factors contribute to
variable steady state levels of oxidative stress which have to be taken into account in
exposure studies.
ROS is a collective term that includes both oxygen radicals and certain nonradicals
(Table 8) that are oxidizing agents and/or are easily converted into radicals (e.g. O3,
ONOO‾, 1O2 and H2O2). All oxygen radicals are ROS, but not all ROS are oxygen
radicals.
● ─
Fig. 4. O2 ,
C
H2O2
C a-Cu
Q (Fe-S)
(Fe-S)
Q (Fe-S)
a3-Cu
FMN FAD
b
H2O ½ O2
NADH Succinate
Hydrogen peroxide (H2O2): Hydrogen peroxide is not a free radical. However, its
● ─
importance lies in the fact that it is able to cross lipid bilayers and like O2 shows
preferential activity towards specific biological molecules e.g. ferritins. It aids in the
formation of hydroxyl radical via oxidation of transition metals. It plays an important role
in phagosomes in ROS mediated microbial elimination as a precursor to various ROS
●
such as HOCl and OH.
● + 2+
Hydroxyl radical ( OH): It is formed from H2O2 by transition metal (Cu /Fe )
catalysed reaction known as the Fenton reaction.
+ 2+ ● ─ 2+ 3+
H2O2 + Cu /Fe OH + OH + Cu /Fe (Fenton reaction) (Reaction 4)
Hydroxyl radical is highly reactive. Its reactions with various biological molecules give
rise to numerous adduction products; DNA adducts being one of them, which may lead
to mutagenesis if not repaired (Nordberg and Arn´er 2006, Halliwell and Gutteridge
2006).
ɤ- carboxyl linkage
Functions of Glutathione:
1. It acts as electron donor keeping the cellular thiols in reduced state.
2. It detoxifies electrophiles by conjugation reactions catalysed by Glutathione-S-
Transferase (GST).
3. Glutathione peroxidases utilize it as an electron donor while reducing H2O2 and
other peroxides. In cytosol and mitochondria where catalase is not present, GSH
plays an important role in eliminating H2O2.
4. Providing a reservoir for cysteine and
5. Modulating critical cellular processes such as DNA synthesis, microtubular-
related processes, and immune function.
Vitamin E: (α-, β-, γ- and δ-) Tocopherols and (α-, β-, γ- and δ-) tocotrienols constitute
the antioxidant group commonly known as Vitamin E. The different forms (α-, β-, γ- and
δ-) differ in their absorption and activity, α-tocopherol being the most effective
biologically. Tocopherols and tocotrienols are similar structurally with a chromanol ring
and a hydrophobic side chain (saturated phytyl chain in tocopherols and unsaturated
side chain in tocotrienols). The hydrophobic side chain allows penetration into the lipid
bilayers. The -OH group on the chromanol group acts as electron donor in reduction of
ROS.
These molecules act as important sinks for ROS that propagate chain reactions of lipid
peroxidation. The tocopheryl radicals formed after the chain breaking reaction can be
regenerated by reaction with ascorbic acid with GSH oxidation or with ubiquinol. When
the tocopheryl radicals are oxidized by radical species resulting into a nonradical
tocopheryl species they are excreted after glucuronic acid conjugation and have to be
replaced by dietary supplementation.
In mammalian tissues, α-tocopherol occurs most abundantly, especially in adipose
tissue, a main storage site. A special protein α- TTP (α-tocopherol transfer protein) with
maximum affinity to α-tocopherol is expressed mainly in liver along with other tocopherol
bonding proteins ensures secretion of α-tocopherol into the circulation for availability to
peripheral tissues. Apart from antioxidant action, vitamin E plays other important roles
such as regulation of enzyme activities (e.g. PLA2 and cox-2). (Herrera and Barbas
2001).
Ascorbic acid or Vitamin C: Vitamin C includes Ascorbic acid its other oxidized forms
such as Ascorbyl radical and dehydroascorbic acid (DHA). Ascorbic acid is a water-
soluble antioxidant that can be oxidized in a two-step reaction.
Loss of one electron to form ascorbyl radical from ascorbate- the ascorbyl radical can be
reduced back by the action of semidehydroascorbate reductase or thioredoxin reductase
or dismutated to Ascorbate and DHA and 2. Ascorbyl radical loses one more electron to
form the fully oxidized dehydroascorbic acid (DHA). DHA is reduced back by
glutaredoxin or thioredoxin reductase. If not reduced to ascorbate, DHA is hydrolysed to
2,3-diketo-L-gluconic acid which is further catabolized to oxalic acid and L-threonic acid.
Vitamin C acts as an important electron donor in a number of reactions, the most
important of which include hydroxylation in collagen synthesis, tyrosine metabolism,
steroid metabolism, nitric oxide synthase activity and regeneration of α-tocopherol from
tocopheryl radical. Ascorbic acid and DHA both have low reduction potential enabling
them to react with almost all of the ROS. However the ascorbyl radical once formed has
very low reactivity avoiding potential reactions with oxygen where a peroxyl radical can
be formed (Higdon and Balz 2002).
Reactive peroxyl radicals, able to react with both membrane proteins and Poly
Unsaturated Fatty Acids (PUFAs), are formed when carbon-centred radicals react
with O2.
2. Singlet oxygen can react directly with PUFAs to form lipid peroxides.
+ 2+
3. Transition metals (Cu /Fe ) accelerate lipid hydroperoxide formation by either
accelerating the formation of hydroxyl radicals from H2O2 or splitting lipid
hydroperoxides to form an alkoxyl and a peroxyl radical through Fenton reactions
(Halliwell and Gutteridge 2006).
None of these reactions are exclusive and multiple types of adducts may be formed
depending upon prevalent physiological conditions.
Hydroxyl radical attack on aliphatic amino acids produces their hydroxylated derivatives
while in addition to hydroxylation, aromatic amino acids give rise to phenoxyl radicals
that can conjugate with other phenoxyl radical (e. g. dityrosine). Methionine is one of the
most easily oxidizable amino acids.
Chain reactions can also occur in protein oxidation in oxidizing conditions where the
initial amino acid oxidation products react with oxygen forming peroxyl radicals. These
radicals then abstract protons producing peroxides and free radicals that participate in
further chain reactions.
JNK-
JNKs are activated by a variety of environmental stresses and exposure to cytokines,
transcription and apoptosis. They are required in inflammatory responses. JNK is
activated by ROS by inactivation of JNK inhibitors (Chen et al 2001; Bernardini et al
2000). The JNK group includes 3 kinase genes, JNK1, JNK2 and JNK3. JNK1/2 have
been studied extensively and are present ubiquitously.
Erk-
Erk1 and 2 constitute the Erk kinases. They are involved in cell differentiation and are
activated by G proteins and RTK receptors through Ras. They provide proliferative
signals to fibroblasts leading to their malignant transformation, proliferation and survival.
p38-
p38 kinase is also activated by environmental stress and immune response. Oxidative
stress response by p38 activation takes place via SRK activation.
COX-2-
Cyclooxygenase catalyses the rate-limiting step in prostaglandin biosynthesis, molecules
mediating inflammatory and immune responses. There are two different isoforms of
COX, COX-1 and COX-2. COX-1 is constitutively expressed and functions as a
housekeeping enzyme. However, COX-2 is induced by mitogens, cytokines and growth
factors of epithelial cells, and is important in prostaglandin production in response to
inflammation (Nishimura et al 2004).
1.9.1.2. In vivo evidence for oxidative stress/damage after areca nut or Pan masala
feeding:
Upon dietary feeding of areca nut or its extracts, effects on antioxidant levels and
antioxidant enzyme activities in hepatic and extrahepatic tissues are not uniform and
differ from study to study. Majority of the studies do not show a drastic decrease in these
parameters. Indeed, some of the studies have noted an elevation (Shivapurkar et al
1978) or no change (Joseph et al 2004) in these parameters, which is possibly
attributable to adaptive response.
Rigorous in vivo testing of biologically important oxidative stress markers such as adduct
measurements upon exposure to either pan masala or betel quid or related ingredients
is lacking except a single report (Chen et al 2002) as according to our knowledge. In this
report the authors observed an elevation in 8-OHdG levels in hamster buccal mucosa
after betel quid feeding for 14 days.
Direct mechanistic evidence in human studies has been restricted to ROS detection
using of o- and m-tyrosine formation as endpoint after addition of L-phenylalanine in the
chew (Chen et al 2006, Nair et al 1995). These experiments provided a direct proof of
ROS generation in physiological conditions that is important considering that ROS
species are generally very short-lived.
There are several factors influencing measurements of oxidative stress parameters on
quid chewing. They include
a. Extent of inflammation if whole areca nut or its pieces are used
b. pH determination if any of the extracts are used
c. Ensuring ROS status at the time of application if extracts are used, as these are very
short-lived species.
These factors should be taken into consideration as possible in the given set of
experimental conditions.
O O•
OH
OH O O‾
+
pH ≥ 9.5 2H
Superoxide dismutase
Semiquinone radical + O2 ● ‾ H2O2 (Reaction 6)
O2
2+ + 3+
H2O2 + Fe + H OH• + Fe +(Reaction
H2O 8)
Fenton’s Reaction
● ‾ + (Reaction 7)
O2 + H2O2 + H
Polyphenols can also act as ROS quenchers by
accepting ROS generated in autooxidation reactions.
Reaction kinetics of polyphenol autooxidation may be different in body tissues and fluids
because H2O2 production in vitro, is dependent on many factors, including media
composition and pH changes (Sang et al 2005, Long et al 2007, Long et al 2000). This
calls for caution while interpreting in vitro genotoxicity testing.
Polyphenols such as tannins also give Pan Masala its astringent taste and colour.
Phenolic groups of polyphenols are in constant equilibrium with the surrounding aqueous
medium (Reactions 8 and 9) with the existence of phenoxide groups (R-O-), which are
the main colouring agents.
1.To investigate if Pan masala exposure leads to oxidative damage in vitro and in
experimental animal models as judged by oxidized DNA, protein and lipid markers.
2.To investigate if Pan masala exposure leads to oxidative stress in experimental animal
models as judged by decrease in the levels of cellular antioxidant(s) and / or activity of
anti-oxidant defense enzymes in tissues of exposed group.
3.To apply validated methods to measure oxidative stress if any in exfoliated and/or
blood cells from limited number of exposed and unexposed volunteers.