Introduction 2

∫Introduction∫

1.1. Cancer as the modern day scourge: Irrespective of the country, region or continent, emergence of cancer has become an important issue in our civilization. It has a large impact on global health by being among the three leading causes of death for adults. It causes more deaths (12.5% of all deaths globally) than HIV/AIDS, tuberculosis and malaria put together (WHO report). Cancer is characterized by uncontrolled cell growth and division in the affected tissue, surpassing normal cell growth and division and spreading to other tissues (metastasis). Causation of cancer itself has multiple routes but essentially it originates in mutations that make it possible for the cell to grow and divide uncontrollably resulting in carcinogenesis. These mutations are mostly brought about by environmental exposure to carcinogens- agents (chemicals or radiations) that cause cancer. The initial malignancy known as a primary tumour can originate in any one of the tissues, including but not exclusive of breast, kidney, prostate, lungs, liver and oral cavity. 1.1.1. Oral cancer and environmental carcinogens: Cancer incidences for the various primary sites vary according to the region (e.g. prostate, breast, lung, colorectal in western hemisphere and oral, lung, cervix, pharynx, breast in South-Central Asia) due to differences in diet and exposure to various substances. Overall, oral cancer is the eighth most common cancer in the world. In developing countries and areas of South Central Asia, cancer of the oral cavity is among the three most frequent types of cancer, associated with prevalence of chewing habits with or without tobacco along with consumption of alcohol and smoking in these areas. The connection of tobacco with cancer is well known but chewing habits without tobacco have also been linked to the incidence of oral cancer. Such habitués consume various mixtures according to personal preferences containing areca nut and/or lime and/or catechu along with various other additives. This habit is widespread and India has the largest population of areca nut consumers (Gupta and Warnakulasuriya 2002). Chewing products without tobacco such as Pan masala (a chewing mixture containing areca nut, catechu and lime but not tobacco) have attracted attention recently because of their popularity. The recent prevalence of these products in India is due to the attractive packaging and aggressive marketing (181% rise in 2004 compared to 2003 in Advertising in Print medium) of a chewing mixture called as Pan masala.

1.2. Pan Masala: This chewing mixture primarily contains Areca nut (70%), Lime (10%), Catechu (1%) and various flavouring agents (Bhisey 2000). It is attractively and conveniently packaged in sachets (Photos of Pan masala sachets and individual ingredients are to be taken in photography department. I already have various Pan masala sachets). It may also contain transition metals (Cu and Fe) and aldehydes such as acetaldehyde, acrolein and propionaldehyde (personal communication with Dr Pakhale). However, it is presumed to be harmless by general public, as it does not contain tobacco or any known/unknown strong carcinogens. 1.2.1. Pan masala components: Areca nut: Areca nut is the seed of the areca, or betel palm (Areca catechu), family Palmae. (Chart 1 lists constituents of the seed and Table 1 enumerates percentage composition of areca nut). It has been classified as a Group I carcinogen by IARC (IARC Monographs, Vol 85). CHART 1 Areca nut constituents Areca nut Polyphenols

Crude fibres

Tannin

Carbohydrates

Fats Protein Saponins Gums Carotene

Mineral Matter

Alkaloids

Flavonoids Anthocyanidine Leucocyanidine

Gallotannic Gallic D-Catechol acid acid

Lauric acid Phiobatanin

Myristic acid

Oleic acid

Calcium Arecoline

Arecaidine

Arecolidine

Guvacine

Sitosterol

Phosphorous

Iron

Isoguvacine

Adapted from Ranadive et al (1976) and Raghavan and Baruah (1958) (All components are not listed.)

Guvacoline

Table 1 Percentage composition of Areca nut as according to ripening stages Sr. No.

Constituents

Green (unripe) nut

Ripe nut

69.4-74.1 17.3-23.0

38.9-56.7 17.8-25.7

1 2

Moisture content Total polysaccharides

3

Crude protein

6.7-9.4

6.2-7.5

4

Fat

8.1-12.0

9.5-15.1

5

Crude fibre

8.2-9.8

11.4-15.4

6

Polyphenols

17.2-29.8

11.1-17.8

7

Arecoline

0.11-0.14

0.12-0.24

8

Ash

1.2-2.5

1.1-1.5

(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 (1333%) 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 Intolerance to spicy food Petechiae Depapillation of tongue Oral ulceration

Fibrous bands Trismus Flattening of palate Hockey-stick uvula Reduced tongue mobility

Leathery mucosa Taste disturbance

Xerostomia 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. Sr No 1

Case reports

Citation

Country

Prevalence of OSF highest in chewers

Saraswathi et al 1990

India

2

Betel quid usage in a hyperendemic area for oral and pharyngeal cancer. Increased prevalence of OSF and oral carcinoma in areca nut chewers Various precancerous conditions associated with areca nut chewing OSF Case report in an Indian migrant

Tovosia et al 2007 Tang et al 1997

Solomon islands China

Chung et al 2005

Taiwan

Reichart et al 2006

Germany

3 4 5

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

No

substance

1 2

Areca Betel quid

Areca Nut

Betel

Catechu

Slaked lime

Leaf

Inflorescence

Stem

without 3 4

tobacco Pan masala Lao-hwa

5

(Taiwan) Betel quid

6

(Taiwan) 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. 1.2.3. Mutagenicity and Carcinogenicity of Pan masala: Following is a brief summary of carcinogenic and mutagenic potential of Pan masala and its ingredients. Bulk of the literature is summarized in Tables 5 and 6.

Table 5 Bioactivity of Pan masala ingredients:

Ingredients Areca nut

Activity Genotoxic activity in human

Literature Stich et al 1981

buccal mucosa and PBL, and

Dave et al 1992

CHO cells Induction of DNA strand

Sundqvist et al 1989

breaks and DNA protein crosslinks in human buccal

Catechu

epithelial cells Carcinogenic activity in mice

Bhide et al 1979, Rao & Das 1989,

and rats. Risk factor for oral precancer

Prokopczyk et al 1987 Chin & Lee 1970, Sinor et al 1990

and cancer Mutagenic activity Dominant lethal mutation in

Stich et al 1983 Giri et al 1987

mice Chromosomal damage in Lime

mouse bone marrow cells Irritation and hyperplasia of

Dunham et al 1966

oral mucosa Generation of reactive oxygen

Nair et al 1992

species with areca nut

Table 6 Bioactivity of Pan masala Mutagenicity in Ames assay Impairment of liver function Sperm head abnormality Cytogenetic damage in human buccal

Bagwe et al 1990, Polasa et al 1993 Sarma et al 1992 Mukherjee et al 1991 Adhvaryu et al 1989,

mucosa and PBL, and in CHO cells Co-carcinogenic activity in mouse skin,

Dave et al 1991 Ramchandani et al 1998

stomach and esophagus Multi-organ carcinogenic activity in mice Association with oral submucous fibrosis Chronic toxicity in mice

Bhisey et al 1999 Anuradha & Devi 1993, Babu et al 1996 Nigam et al 2001

1.2.3.1. Genotoxicity and mutagenicity: Mutagenic potential of Pan masala extracts varies with solvents used. Various extracts of Pan masala as well as quids prepared in labs have been tested for their genotoxicity in bacterial and mammalian assay systems. In Ames assay, aqueous, aqueous: ethanolic, ethanolic extracts and their nitrosated mixtures (nitrosation reactions at acidic pH) of both Pan masala and laboratory prepared quid were used to test for mutagenicity and dose response. In the absence of metabolic activation, only ethanolic extract was weakly positive (Bagwe et al 1990). In an independent study, the aqueous extract was mutagenic only at the higher dose levels of 100 µg/plate (Polasa et al 1993). As compared to bacterial test systems, Pan masala shows a more pronounced effect in mammalian assays. DMSO and aqueous extracts of pan masala show increased frequency of chromosomal aberrations and sister chromatid exchanges in CHO cells (Patel et al 1994, Trivedy et al 1994). Addition of betel leaf to pan masala and pan masala with tobacco extracts induced dose dependent decrease in both CAs and SCEs. On exposure to increasing concentrations of betel leaf, levels of genotoxic markers increased. Opposing effects of betel leaf extract depending upon concentrations used, indicate complex nature of chemical reactions taking place in chewing of areca nut containing mixtures (Trivedy et al 1994).

1.2.3.2. Carcinogenicity, tumour promotion and other biological activities of Pan masala in mammalian systems: Lifetime exposure of powdered Pan masala through diet was carcinogenic to Swiss mice (7 out of 108 animals at 5% dose level) (Bhisey et al 1999). In a similar study Nigam et al reported increased incidence of tumours in various organs of Swiss mice after feeding Pan masala (2%) for 56 weeks. However in this study, authors did not provide statistical evaluation of their results. Investigations using initiation/promotion specific protocols by ethanolic pan masala extract (EPME) painting in hairless mice indicated that pan masala

primarily acts as a tumour promoter (Ramchandani et al 1998). Increase in epithelial tumor incidence rate after DMBA initiation/EPME promotion protocol over to that of the respective control was statistically significant at 25 mg dose after 40 weeks of EPME exposure. DEN initiation/EPME promotion protocol registered a statistically significant increase in oesophageal tumor incidence and rate at 25 and 50 mg dose level after 3 months of EPME exposure. 1.2.3.3. Development of precancerous lesions and Pan masala chewing - Human studies: Human epidemiological studies have confirmed the relationship between Pan masala chewing and precancerous lesions. Pan masala chewers run a higher risk of developing OSF than non-chewers (Hazare et al 1998). Also Pan masala chewers developed OSF after shorter duration of the habit (2.7 ± 0.6 yr) as compared to other chewing habits (8.6 ± 2.3 yr) (Shah and Sharma 1998) indicating accelerated disease process. 1.2.4. Active elements in Pan masala: In spite of the conspicuous bioactivity, Pan masala has not been shown to contain any known/unknown strong carcinogen. Therefore other entities such as formation of reactive oxygen species (ROS) (formed after autooxidation of areca nut extracts at pH ≥ 9.5) and areca nut specific nitrosamines (ASNAs) (e.g. 3-Methylnitrosaminopropionaldehyde (MNPA), 3-Methylnitrosaminopropionitrile (MNPN) and various aldehydes present in Pan masala mixture have been proposed to play a role. In vitro evidence for oxidative stress after Pan masala exposure is encouraging (Nair et al 1987, Bagachi et al 2002, Yi et al 1990, Liu et al 1996). Production of ROS and subsequent oxidative stress and damage seems particularly to be an attractive hypothesis as these factors have been known to play a role in various disease processes such as carcinogenesis, aging and tissue degeneration. 1.3. ROS and disease processes: Reactive oxygen species (ROS), highly reactive intermediates of oxygen including radicals and other molecular species (e.g. superoxide and peroxide ions), are also formed in normal physiological processes such as mitochondrial phosphorylation and therefore background levels of oxidative damage always exist in cells (Halliwell 1999). They are also formed in pathological conditions such as inflammation. These ROS if not

quenched sufficiently by cellular antioxidant mechanisms oxidize cellular molecules; DNA, proteins and lipids to form harmful products and affect cell signalling pathways. This damage to cells caused by oxidants, or chemicals that capture electrons from other substances is termed as oxidative damage. Whereas Oxidative stress is defined as a disturbance in the prooxidant-antioxidant balance in favour of the former, leading to potential damage (Fig 1) (Sies et al 1991). Accumulation of oxidative adducts formed after addition of oxygen atom/s to biomolecules and/or other products of oxidative reactions may lead to pathological consequences; e.g. Carcinogenesis, Alzheimer’s disease, Rheumatoid arthritis, etc ( Karihtala and Soini 2007, Dalle-Donne et al 2003). This accumulation increases with age as repair systems of the body fail to recognize and/or eliminate the damaged molecules. It is not a surprise then that incidence of pathological conditions associated with oxidative damage increases with age. Fig. 1

A N T I O X I D A N T

P R O O X I D A N T

ROS play an important role in carcinogenesis by, causing mutations in cell cycle

Oxidative damage regulating genes and affecting cell signalling pathways, ultimately 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

Early biological lesion

Protein adducts

Allelic loss or gain Chromosomal aberration Gene mutation

Lipid peroxidation

Table 7 Pathological conditions in which levels of 8-OHdG have been measured. Organ syste m Blood

Disease

Comments

Blood Acute lymphoblastic leukemia (ALL)

Lymphocyte DNA lesion levels significantly (P < 0.05) elevated in ALL vs. control subjects (Stenturker et al 1997).

Brain

Alzheimer’s disease(AD) Multiple sclerosis

Higher levels of 8-OH-dG in cortex and cerebellum of AD patients vs. controls (Lezza et al 1999). Significantly elevated levels of 8-OH-dG in plaques, compared to normal-appearing white matter in multiple sclerosis-affected cerebella (Vladimirova et al 1998). Breast Invasive ductal Significantly elevated levels of 8-OH-dG (P <0.001) carcinoma in malignant breast tissue; also levels significantly greater (P < 0.007) in estrogen receptor-positive (ORP) vs. ORP-negative malignant tissue (Mussarrat et al 1996). Cardiovascular Strong association (r = 0.95, P < 0.01) between disease premature coronary heart disease in men and lymphocyte 8-OH-dG levels (Collins et al 1998). Colon Colorectal Significantly elevated levels of 8-OH-dG (P <0.005) cancer (CRC) in tumor tissue compared to normal mucosa (Oliva et al 1997). Liver Hepatoblastoma Positive immunohistochemical staining for 8-OH-dG in liver sections from all 5 patients with hepatoblastoma (Ikeda et al 2001). Chronic Positive immunohistochemical staining for 8-OH-dG hepatitis, in all diseased liver sections; no staining in control alcoholic liver liver sections (Kitada et al 2001). disease, primary biliary cirrhosis. Lung Cystic fibrosis Urinary levels of 8-OH-dG significantly raised vs. control subjects. Cooke et al 2002 Lung cancer Lymphocyte DNA levels of 8-OH-dG significantly elevated (P < 0.05) compared to controls (Vulimiri et al 2000). Skin Atopic Urinary 8-OH-dG significantly higher than in dermatitis controls (P < 0.0001) and correlating with disease severity index. Cooke et al 2002 Diabetes Patients with both type I and II diabetes had mellitus Type I significantly higher levels of urinary 8-OH-dG, and II compared to controls (Krapfenbauer, et al 1998). Rheumatoid Levels of urinary 8-OH-dG significantly elevated (P < arthritis 0.001) compared to control subjects (Lunec et al 1994). (Adapted from Cooke et al 2003)

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.

1.5. Reactive oxygen species, oxidative damage and oxidative stress: Oxidative stress and resulting damage during carcinogenesis is caused by ROS (e.g. superoxide and peroxide ions) that are produced mostly during metabolism of the carcinogen and inflammation. ROS are produced by single electron transfers (at a time) to and from the molecular oxygen. However, almost all forms of life depend on oxygen for efficient energy production. So how does this lifesaving gas become toxic to cells? 1.5.1. Formation of ROS from molecular oxygen: ROS are toxic because of their high reactivity mostly due to the presence of unpaired electrons. Any species capable of independent existence that contains one or more unpaired electrons is called a free radical. The O2 molecule is a free radical as it has two unpaired electrons. The two electrons in O2 have the same spin. This is the most stable state, or ground state, of O2 (Halliwell and Gutteridge 2006). Acquiring energy to release the spin requirement forms more reactive forms of O2. These are singlet oxygen species (1O2). Oxygen is, thermodynamically, a potent oxidizing agent able to react via single electron transfers. Single electron transfers from the ground state produce superoxide and peroxide ions during reactions with nonradicals/radicals (Fig. 3). When O2 accepts a single electron it becomes a superoxide radical O2

● ─

. Addition of another electron to superoxide radical results in peroxide ion.

Two more electrons added give O2 ̶ (oxide ions) resulting in formation of water (Reactions 1 and 2).

σ*2p π*2p π 2p σ 2p σ*2s σ 2s σ*1s σ 1s Groundstate O2 3 Σg─ O2

Singlet O2 1 Δg O2

Superoxide O2● ─

Peroxide O22 ─

Singlet O2 1 Σg+O2

Fig. 3 Electron states of diatomic oxygen molecule and its derivatives. The oxygen atom has eight electrons and O2 has 16 electrons. Adapted from Halliwell and Gutteridge (2006).

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. Table 8 ROS molecules Free Radicals 1.Superoxide, O2 · ̶

1.H2O2

2.Hydroxyl, OH·

2.Ozone, O3

3.Hydroperoxyl, HO2· (protonated

3.Singlet oxygen (O21∆g)

superoxide)

4.Organic peroxides, ROOH

4.Carbonate, CO3

Nonradicals

·̶

5.Peroxynitrite, ONOO2

5.Peroxyl, RO2 ·

6.Peroxynitrate, O2NOO2

6.Alkoxyl, RO·

7.Peroxynitrous acid, ONOOH

7.Carbon dioxide radical, CO2 1 2

+

·̶

8.Peroxomonocarbonate, HOOCO2

8.Singlet O ∑g Adapted from (Halliwell and Gutteridge 2006)

In aerobic cells toxic ROS production is assuaged by single electron transfers along the electron transfer chain sequestering the reactive intermediates till complete reduction of one oxygen molecule to water in mitochondrial membrane (Fig. 4). For complete reduction of one oxygen molecule, 4 electrons are converted into two molecules of water (Reactions 1 and 2). Cytochrome oxidase complex in mitochondria reduces oxygen molecules by adding electrons one by one. This is so because of the inherent inability of the oxygen molecule to react with two paired electrons of a nonradical in a single reaction. O2 has same spin unpaired electrons, which will not fit an electron pair of opposite spins. Mitochondria reduce 95% or more of O2 to water. Despite efficient electron transfers, aerobic cells are always exposed to a small amount of ROS escaping from the mitochondria (e. g. ~10-8 M in rat liver cytosol) (Boveris and Cadenas et al 1997). These escaping ROS and reactive moieties from metabolism of xenobiotics render the cell susceptible to oxidative damage.

Two-electron reduction (plus 2H+ ) of oxygen: O2

H2O2 (protonated form of O22‾)

(Reaction 1)

2H2O (protonated form of O2‾)

(Reaction 2)

Four-electron reduction (plus 4H+ ) O2

● ─

Fig. 4.

O2 H2O2 (Fe-S)

,

C C

Q

Q

(Fe-S)

a-Cu

(Fe-S)

FMN

FAD

a3-Cu

b

NADH

Complex I

Succinate

Complex III

Complex II

H2O ½ O2

Complex IV

Mitochondrial respiration chain. Complex I – NADH-ubiquinone oxidoreductase, Complex II – Succinate dehydrogenase, Complex III - ubiquinol-cytochrome c oxidoreductase, Q – semiquinone, a-Cu/a3-Cu/b/C1 – cytochrome a/a3/b/c Superoxide and hydrogen peroxide are thought to originate during semiquinone formation at complex I and II.

1.5.2. A brief overview of biologically important ROS: We will take a closer look at some of the reactive molecules described above which play an important role in cellular processes. Superoxide radical (O2

● ─

): Superoxide radical lacks the ability to cross lipid

membranes and mostly remains confined to the compartment where it was produced. The most important site for O2

● ─

production in aerobic cells is the mitochondrial

membrane where leakage of electrons from the electron transport chain and subsequent reaction with O2 forms O2

● ─

(Karihtala and Soini et al 2007). It reacts with very few

molecules in vivo and hence is deemed not to be very reactive. But it can react with ironsulphur clusters in enzymes ultimately releasing iron and inactivating the enzyme. It can ●

also react with Nitric oxide (NO ) to give the highly reactive peroxynitrite radical −

(ONOO ) (Reaction 3). Such few but biologically important reactions make superoxide radical elimination highly desirous. NO

●

+ O2●

─

−

ONOO (Peroxynitrite radical)

(Reaction 3)

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.

+

H2O2 + Cu /Fe

2+

●

─

2+

OH + OH + Cu /Fe

3+

(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). 1.5.3. Oxidative damage: Thus various ROS alter structure and function of the molecules that they react with and induce damage to most of the cellular biomolecules. DNA damage leads to transcription errors and mutagenic lesions, protein damage to ultimately form non-functional protein aggregates and lipid damage leading to leaky membranes and formation of toxic products. Most of these molecules are nonradicals. A radical reaction with the biological nonradicals may have any one of the several fates and result in oxidative damage. •Adduction: addition of the radical to the nonradicals will yield an adducted molecule. Examples include addition of the OH• radical to DNA bases (8-OHdG, 8-hydroxy-2’ – deoxyadenosine, Thymine glycol, etc.), to amino acid side chains (2′- oxo - histidine) and to lipids. •Reduction: a radical cation may add an electron to a nonradical reducing it to a radical with an unpaired electron and hence turning it into an oxidizing agent. •Oxidation: a radical may snatch an electron to reduce itself while oxidizing the electron donor. •Formation of carbon centred radicals and chain reactions: a radical sometimes may break a C-H bond by taking the hydrogen atom, leaving behind a carbon centred radical. The carbon-centred radicals react fast with oxygen to yield peroxide radical. Peroxide radicals will again break a C-H bond and so on and so forth the chain reaction will continue generating more and more radicals. These reactions occur most commonly in membrane lipids forming lipid peroxides.

1.5.4. Oxidative stress and Antioxidant defences:

To protect themselves from the recurring damage from ROS arising from endogenous (mitochondrial leaking) and/or exogenous sources (xenobiotics, UV radiation, etc.), cells have evolved various antioxidant defences. Highly efficient electron transfer chains have evolved to reduce endogenous ROS production. In addition, proteins involved in the process are compartmentalized reducing exposure of other cellular components to the harmful radicals and contain the damage caused to a particular compartment. To further reduce concentration of oxygen radicals, cells employ ROS scavengers. An army of specialized enzymes and low molecular weight antioxidants capable of reducing ROS comprising of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), peroxiredoxins (Prx) and glutathione (GSH), ascorbic acid, etc is present in the cell. Many of these enzymes and antioxidants show increased activity and/or levels upon exposure to ROS.

1.5.4.1. Antioxidant enzymes: Superoxide dismutase (SOD): Superoxide dismutase catalyses the dismutation of O2•¯ to hydrogen peroxide and O2. The reaction catalysed by SOD is extremely fast limited in essence only by the rate of diffusion. (Nordberg and Arn´er 2001). Superoxide radical elimination in vivo is important because of the large amount of O2•¯ formation in mitochondria and by enzymes such as xanthine oxidase during ischemia-reperfusion (Nordberg and Arn´er 2001). SOD is present in cytosol, nucleus, mitochondria and extracellular compartments (Slupphaug et al 2003). Depending on the metal cofactor used by the enzyme, SODs present in eukaryotic animal cells can be categorized into Mn SOD (in mitochondria) and Cu/Zn SOD (cytosolic). Of these, mitochondrial SOD is inducible (Nordberg and Arn´er 2001). Catalase and Glutathione Peroxidases (GPx): Catalase and glutathione peroxidases dismutate hydrogen peroxide to water and molecular oxygen. ♦ Catalase: Catalases are mainly heme containing peroxisomal enzymes. Their catalysis reaction is extremely efficient and cannot be inhibited by any concentration of H2O2 (Curtin et al 2002). Apart from this, they also function in detoxifying other substrates such as phenols and alcohols via coupled reduction of H2O2 (Nordberg and Arn´er 2001). ♦ Glutathione peroxidases: As opposed to peroxisomal catalase, mammalian glutathione peroxidases are cytosolic selenocysteine containing enzymes and work in conjunction with another small molecule antioxidant, glutathione by oxidizing it to GSSG,

the disulfide form while reducing H2O2 and other peroxides such as lipid peroxides. GPx enzyme activity is important by virtue of its ability to reduce lipid peroxides. They are also shown to serve a structural role in specific physiological conditions (Ursini et al 1999). Peroxiredoxins: As compared to glutathione peroxidases, peroxiredoxins (Prx or thioredoxin peroxidases) are enzymes capable of directly reducing peroxides, both hydrogen peroxide and different alkyl hydroperoxides, without glutathione reduction. Glutathione-S- Transferases (GST): GSTs are major detoxifying enzymes found mainly in the cytosol. They represent a highly diverse enzyme superfamily classified according to species. 7 species independent classes exist ((alpha, kappa, mu, pi, sigma, theta, and zeta) on the basis of their amino acid sequence, substrate specificity and Immunological properties. They catalyse conjugation of electrophilic substrates to GSH. They also have peroxidase and isomerase activities and can inhibit Jun kinases. Oxidation of GST protein inactivates it by forming a disulfide bridge between cysteine 47 and cysteine 101 (Lo Bello et al 1998). These antioxidant enzymes reduce ROS by oxidizing several substrates particularly smaller molecules known as low molecular weight antioxidants. Their oxidation may be reversible (e.g. GSSG) or irreversible (ascorbate). The oxidized forms have very low reactivity and serve as the final step in the ROS reduction process.

1.5.4.2. Low molecular weight antioxidants: Glutathione system: Glutathione is the most abundant intracellular (90% in cytosol and 10% in mitochondria) thiol-based antioxidant. It is a tripeptide, ɣ-glutamylcysteinyl glycine (Fig. 5).

SH O

NH2

CH2

O

C CH CH2 CH2 C NH CH C NH CH2 C HO

O

O

OH

ɤ- carboxyl linkage

ɤ-glutamyl

cysteinyl

glycine

Fig. 5 The structure of glutathione, ɣ-glutamylcysteinyl glycine. The amino-terminal glutamate and cysteine are linked by the ɣ-carboxyl group of glutamate. It exists in two forms, the reduced form, GSH, and the oxidized form, GSSG. Under normal conditions GSH accounts for almost 95% of the total glutathione pool. It provides reducing equivalents to proteins, GSH-mixed disulfides and low molecular weight compounds and performs various functions in conjunction with other antioxidant systems (Fig. 6) (Lu 1999).

Fig. 6 Major glutathione-associated antioxidant systems. [adapted from Nordberg and Arn´er (2001)] 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-STransferase (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, microtubularrelated 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 watersoluble 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). 1.6. Cellular damage due to oxidative stress: If the antioxidant systems mentioned above are not able to contain ROS electrophilic attacks on most of the cellular macromolecules take place including lipids, DNA and protein. Mechanism of reactions and products vary for each of these. Lipid oxidation reactions are fascinating to study with chain reactions being a hallmark. 1.6.1. Lipid damage: In the initial reactions of lipid oxidation, a reactive oxygen species molecule such as a hydroxyl radical or singlet oxygen abstracts a single hydrogen atom from the C-H bond. This gives rise to lipid radicals, which in turn attack more C-H bonds constituting a chain reaction. 1.6.1.1. Lipid oxidation reactions: The initial lipid radicals can be formed in multiple ways1. When oxygen radicals react with membrane lipids, they abstract a hydrogen atom from a C-H bond to form carbon-centred radicals. C-H + OH

•

•

C + H2O •

(NO2 )

(Reaction 5)

(HNO2)

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).

1.6.1.2. Lipid oxidation and its biological effects: A single initiation reaction forming peroxyl radicals thus, has the potential to generate multiple lipid hydroperoxides through chain reactions in which peroxyl radicals attack the nearest C-H bond. These attacks break down longer PUFA chains into smaller fragments (3-9 carbons in length) ultimately compromising membrane structure. Continuous lipid damage finally leads to a loss of membrane integrity by increasing the membrane fluidity. The cellular membranes leak and in some cases can rupture, compromising cell compartmentalization. It can also produce signalling molecules such as isoprostanes. Lipoxidation by transition metals produces toxic molecules like malondialdehyde, 4-hydroxynonenal (4-HNE), epoxides and hydrocarbons. These molecules can inactivate enzymes and receptors. 4-HNE and malondialdehyde cause DNA damage by forming adducts. These aldehydes are also relatively stable and can diffuse. The smaller fragmented aldehydes formed by breakdown of larger PUFA chains can be grouped on structural basis into, 2-alkenals, 4-hydroxy-2-alkenals, and ketoaldehydes. 2-Alkenals: They are highly reactive aldehydes containing two electrophilic reaction centres (carbon 1 or 3). 4-hydroxy-2-alkenals: They are major products of lipid peroxidation and their accumulation leads to many detrimental cellular effects, e.g. 4-hydroxy-2-nonenal. Ketoaldehydes: Examples in this class include Malondialdehyde (MDA), glyoxal and 4-oxo-2-nonenals. MDA is the most abundant lipid-peroxidation specific aldehyde resulting from lipid peroxidation. These aldehydes can react with and inactivate proteins by formation of a Schiff base at the carbonyl group or Michael addition to the protein followed by cyclization. Adducts formed from these reactions lead to impaired enzyme activity and increased immunogenicity. These α, β-unsaturated aldehydes (enals), e.g. crotonaldehyde, acrolein, malondialdehyde and 4-HNE, induce DNA damage through formation of exocyclic DNA

adducts. Reactions of purine and pyrimidine bases with more reactive epoxy derivatives of aldehydes also lead to formation of these adducts. Malondialdehyde is another lipid peroxidation derived aldehyde that forms addition products on reactions with deoxyadenosine, deoxyguanosine and deoxycytidine. MDA reacts with DNA to form adducts of dG, dA and dC. MDA is the most mutagenic of all lipid peroxidation products and HNE is the most toxic (Esterbauer et al 1990) with effects on expression of cell cycle and apoptosis regulating genes. The reactivity of enals toward dG decreases with the increasing chain length acrolein > crotonaldehyde > HNE (Pan and Chung, 2002). 1.6.2. DNA damage: 1.6.2.1. Generation of DNA Adducts: Along with lipid oxidation products, direct ROS attacks on DNA modify the base/ sugar/ phosphate backbone structure causing erroneous transcription or replication. Because of the hereditary nature of these changes, DNA represents a biologically important ROS target. •

Of the ROS milieu, reactions of the highly reactive hydroxyl radical (OH ) with DNA are well documented. However the species is extremely reactive to diffuse through the nuclear envelope without reacting with cellular components. Therefore, diffusion process has been visualized as occurring in the form of H2O2, which then forms hydroxyl radicals in Fenton reaction in the vicinity of DNA. Hydroxyl radical reacts at multiple sites, addition at double bonds of DNA bases and abstraction of an H atom from the methyl group of thymine and C-H bonds of 2 ´-deoxyribose. Addition and abstraction reactions with DNA bases result in adduct radicals that give rise to different products such as •From addition to pyrimidines Cytosine glycol, thymine glycol (in the presence or absence of oxygen), 5-hydroxymethyluracil, 5-formyluracil, 5-hydroxy-5-methylhydantoin, isodialuric acid and alloxan (in the presence of oxygen), 5-hydroxy-6-hydropyrimidines and 5-hydroxy-5-hydropyrimidines (in the absence of oxygen), •From addition to purines – 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-Gua), 4,6-diamino-5formamidopyrimidine (Fapy-Ade) (In the presence or absence of oxygen),

8-oxo-7,8-dihydroguanine (8-oxodG) (In the presence of oxygen), 2-Hydroxyadenine, Imidazolone, oxazolone. •By abstraction of hydrogen in thymine 5´-hydroxymethyluracil and 5´-formyluracil (in the presence or absence of oxygen). Abstraction of hydrogen from ribose sugar moieties leads to single strand breaks and multiple attacks generate double strand breaks. •

•-

In addition to OH , peroxynitrite radical (OON ) can also cause significant DNA damage resulting primarily in 8-OHdG and 8-nitrodG (8-Nitroguanosine) (Burney et al 1999). None of these reactions are exclusive and multiple types of adducts may be formed depending upon prevalent physiological conditions. 1.6.2.2. Biological effects of oxidative DNA modification: These adducts affect DNA replication and transcription in multiple ways.8-OHdG is one of the most studied oxidative lesion of DNA. 8-OHdG lesions cause G

T

transversions when formed after incorporation into DNA strand. This is due to reversion of the modified base to the energetically favourable conformation in single strands. 8OHdG prevents normal G-C pairing during replication or transcription. 8-OHdA formation may result in A

C substitution. Misincorporation of 8-OHdGTP from free

nucleotide pool opposite of dA results in A

C transversions.

Presence of thymine glycol blocks DNA replication. Microsatellite instability, a common feature of human cancer cells has been shown to occur after exposure to oxidizing agents in cell lines (Turker et al 1999). Presence of 8-OHdG in promoter elements can affect transcription factor binding (Ghosh and Mitchell 1999).

1.6.3 Oxidative protein damage: Proteins are important ROS targets because of their quantity, ubiquity and as molecules governing biological catalysis. They scavenge majority of the ROS (50-75%) (Dalledonne et al 2006). Generation of carbonyl groups on amino acid side chains is one of the most commonly studied and used markers for protein oxidation.

1.6.3.1. Generation of protein carbonyls: Protein carbonyls may be generated by oxidation of several amino acid side chains by formation of ketone as well as aldehyde groups (e.g. in Lysine, Arginine, Proline, and Threonine). Formation of Michael adducts between Lys, His, and Cys residues and lipid oxidation end products such as 4-HNE and MDA introduces carbonyl groups into proteins. They can alter receptor binding and increase aggregation (reaction of 4-HNE Apolipoprotein B at low and high concentrations, respectively). MDA reacts mainly with Lysine residues by Michael addition. HNE is much more reactive to proteins than to DNA. These protein adducts formed by reaction with lipid adducts are known as ALEs (Advanced Lipoxidation End products). Another pathway for formation of protein carbonyls is through glycation/glycoxidation of Lys amino groups, forming advanced glycation end products. 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. 1.6.3.2. Biological effects of oxidative protein modification: Such radical entities also exist in the active state of most of the enzymes, participating in the catalytic reactions. These radicals upon reaction with ROS can lead to protein backbone cleavage and ultimately to enzyme inactivation. Oxidative metabolism enzymes can self-inactivate when exposed to their catalysis intermediates/products e.g. lipid hydroperoxide radicals from lipoxygenase reactions can inactivate the enzyme in the presence of iron. In oxidizing environments, vicinal -SH groups conjugate to form disulfide bonds (S-S) to form inter- and intra-molecular links changing protein conformation.

Depending on the extent of oxidation, hydrophilicity and/or proteolytic susceptibility, tertiary and quaternary structures, immunogenicity and catalytic activity of the damaged protein are altered.

1.7. Oxidative stress biomarkers: Levels of all of the above mentioned ROS, antioxidants, antioxidant enzyme activities and oxidative damage products are utilized to measure oxidative stress. However, each class inherently deals with different aspects of oxidative stress (Table 9) and hence none of them when measured individually will give an accurate picture. ROS themselves can be measured directly however they are shortlived and are difficult to measure in vivo. A group of endpoints belonging to the two groups, 1. Level of oxidative stress in the cell or antioxidant levels and antioxidant enzyme activities and 2. Level of oxidative damage sustained by the cell or levels of oxidative products can be used as biomarkers. Table 9 Classes of oxidative stress biomarkers: Sr

Class of

No

Biomarkers

1

ROS

Advantages

Disadvantages

A direct measure of

Short lived, reactive species hence

exposure

difficult to capture and measure Does not estimate load delivered to

2

3

Antioxidant

Parameter for

target organ Does not measure biological effects

levels/ anti-

biological response to

of oxidative stress

oxidant enzyme

exposure

activities Oxidative

Ease of measurement Direct measure of

Endogenous levels present a

damage

biologically effective

confounding factor

dose 1.7.1. Oxidative stress levels: Oxidative stress can be measured by measuring the antioxidant levels and antioxidant enzyme activities. Enzyme activity is a good measure of the enzyme’s role in vivo as, activity measures biological effectiveness of the enzyme, as at a given time only a

portion of the enzyme may be active as enzymes may get inactivated when exposed to large amounts of ROS. Normally depletion in these levels/activities is taken as a measure of oxidative stress. However expression of most of these molecules is tightly regulated and is induced upon exposure to oxidants. Hence, increased antioxidant levels on mild ROS exposure and depletion upon severe oxidative stress can be expected. Taken at a single time point they provide a single snapshot at cellular events. Continuous monitoring therefore is recommended to get an accurate picture of events. It must be stressed here that oxidative stress and oxidative damage differ from each other greatly. In a cell, oxidative stress may occur without oxidative damage if the cell is able to overcome the ROS exposure by utilizing its antioxidants. This depletes the antioxidant reservoir and ultimately if the ROS exposure continues, cellular components also are affected. Antioxidant enzyme activities and antioxidant levels measured thus give a measure of the load that the cell experiences on its antioxidant reservoirs therefore these can be called as surrogate biomarkers of oxidative stress. Levels of GSH, ascorbic acid, thioredoxin and activities of catalase, superoxide dismutase, etc antioxidant enzymes are some of the most common markers for oxidative stress. Most of these can be measured spectrophotometrically with ease of detection. 1.7.2. Oxidative damage: Oxidative damage products are a direct measure of the biologically effective dose of ROS. There have been numerous attempts to use any one of the oxidative damage products mentioned above as biomarkers but none of them can be cited as the gold standard for measuring oxidative stress. A variety of reasons exist which include that a single ROS species can attack at various positions of the same biomolecule that results in a variety of products. These multitudes of products may then undergo a variety of reactions depending upon the reaction conditions. This variety then makes it impossible to determine the exact ratio of any product present after oxidative stress and relate it to an exact level of exposure. Also when more than one ROS is suspected to be at play, its target molecules may be different and so ideally speaking damage to all the cellular macromolecules should be measured. Also many of these damage products are produced in normal physiological functions of the cell hence it is important to differentiate between the background levels from the exposure levels.

In these, 8-OHdG is measured very frequently because of its ease of detection. However there are many contentions against its use the main being that most of the methods used for its detection cause artifactual generation of 8-OHdG and levels can vary tremendously depending on the methodology used and tissue analysed (145.25 to 0.07 adducts/106 nucleotides as measured by GC/MS/SIM and HPLC/EC respectively) (Bont and Larebeke 2004). Hence its detection needs to be complemented with different methodological approaches. Protein carbonyls are frequently measured by a variety of methods such as western blotting, immunohistochemistry, spectrophotometric measurements, etc. Extent of lipid peroxidation can be measured by evaluating levels of one or more of the numerous products formed after lipid oxidation. However these measurements may not reflect levels of rest of the lipid oxidation products and hence may be incomplete. Measuring lipid hydroperoxides may partially overcome this problem by measuring initial products of lipid oxidation i.e. lipid hydroperoxides rather than products formed at the end (e.g. MDA).

1.7.3. Criteria for the ideal biomarker of oxidative damage: An ideal oxidative stress/damage biomarker should fulfil the technical criteria given below (i) The biomarker should detect a major part, or at least a fixed percentage, of total ongoing oxidative damage in vivo. (ii) The coefficient of variation between different assays of the same sample should be small in comparison with the difference between subjects. (iii) Its levels should not vary widely in the same subjects under the same conditions at different times. (iv) It must employ chemically robust measurement technology. (v) It must not be confounded by diet. (vi) It should ideally be stable on storage, not being lost, or formed artifactually, in stored samples. Although many oxidative stress biomarkers corresponding to one or more of the above attributes have been utilized, none has been able to fulfil all of them. And no one individual marker can be cited as indicative of the extent of oxidative stress/damage

suffered by the system (in vitro/vivo) and consequently measurement of oxidative stress/damage is not without its pitfalls. This is because•ROS production is a natural phenomenon even in the context of normal physiological processes. Hence it is difficult to differentiate between pathological/abnormal and background/normal levels of ROS/oxidative damage. ●

•ROS themselves have extremely short half-life (e.g. O2•¯ - 2-4 µs, H2O2 - 1ms, OH – less than 1µs) (Pessarakli 2005) and very reactive making measurement of ROS production very tricky. •They also lead to a plethora of reactive intermediates and products in varying stoichiometry depending upon reaction conditions. Therefore, levels of any one product cannot be correlated to overall ROS production. •Many oxidatively modified molecules are very similar to their precursor molecules requiring extensive analysis of their properties and instrumentation to differentiate between the two. Because of these reasons, a battery of oxidative stress biomarkers is normally used to determine the amount of ROS production and the extent of oxidative challenge (Halliwell & Whiteman 2004).

1.8. Kinase levels as response markers: Cells respond to stresses such as oxidative stress by mounting defensive responses through various phosphorylating enzymes, mainly, MAP kinases (Mitogen Activated Protein kinases). Jun kinases, JNKs (c-Jun NH2-terminal Kinase), ERKs (Extracellular signal-regulated kinases), p38 kinases are main classes of MAP kinases. Each of these is involved in a specific pathway that interacts with a host of other kinases to bring about stress response. ERKs respond mainly to phorbol esters and growth factors and are involved in the proliferative response. JNKs and p38 kinases are more responsive to stresses such as radiation and osmotic shock and participate in apoptosis during stress. JNK levels are increased in response to increased ROS production by dissociation of GSTπ from JNK thereby activating JNK, by activation of its regulators and by downregulating JNK inhibitors (Benhar et al 2002). Various tumor promoters have been shown to modulate kinase activities and affect cell signalling. They can also induce inflammation by inducing Cyclooxygenase-2 (COX-2) levels.

Levels of COX-2 and levels of phosphorylated kinase molecules i. e. active form of the enzyme are used as response markers to measure biological response to toxicological challenges.

JNKJNKs 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. ErkErk1 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. p38p38 kinase is also activated by environmental stress and immune response. Oxidative stress response by p38 activation takes place via SRK activation. COX-2Cyclooxygenase 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. Oxidative stress due to Pan masala exposure and Polyphenols: In vitro and in vivo studies have shown formation of ROS and in some cases, subsequent oxidative stress/damage after exposure of cultured cells and laboratory animals to areca nut or its extracts. Pan masala extracts have been shown to induce formation of ROS in cultured cells. 1.9.1. Oxidative stress after Pan masala exposure: 1.9.1.1. In vitro exposure to areca nut and Pan masala extracts:

Some of the oxidative stress biomarkers mentioned above, such as 8-OHdG, have been evaluated after Pan masala exposure in vitro. These observations have provided a premise based upon which further studies can be carried out. They also suggest that polyphenols in Pan masala components play an important role in ROS generation after Pan masala exposure. Bagachi et al (2002) described ROS generation upon pan masala exposure in normal human oral keratinocytes with aqueous extract of pan masala. These and other such papers (Yi et al 1990) Liu et al (1996) demonstrated ROS generation and elevation of 8hydroxy-2’-deoxyguanosine (8-OHdG, a marker for oxidative DNA damage) levels in cultured cells after exposure to areca nut containing test substances. However, first evidence of the link between ROS production and autooxidation of areca nut extracts was provided by Nair et al in 1987. They demonstrated for the first time superoxide and hydroxyl radical generation for areca nut extract at alkaline pH (≥9.5, attainable in areca nut chewer’s saliva after addition of lime). Of various areca nut extracts and components tested, tannins gave the highest peak for superoxide production. Superoxide and total ROS production by complete areca nut extract was less when compared to that of the isolated components. This indicates the existence of either ROS quenching species in the mixture or antagonistic reactions between the components resulting in decreased ROS production. Saliva itself inhibited ROS production due to its inherent buffering capacity and the presence of various proteins. Analysis of exogenously added transition metals indicated that presence of iron ions significantly enhances superoxide ion production (Nair et al 1987). In this respect, plant phenolics, present in almost every quid ingredient, are important as they can act in ROS generation as well as quenching of ROS. Various additives can also modulate ROS production during betel quid chewing. (Chen et al 2006) (Jeng et al 1994). Glutathione (GSH, a free thiol which undergoes oxidation and subsequent reduction reactions to reduce cellular ROS) is depleted in oral fibroblasts treated with Arecoline. This decrease is not accompanied either by GSSG elevation (oxidized form of GSH) (Sundqvist et al 1989) or ROS production (Chang et al 2001). Reactive molecules other than ROS can directly react with GSH and form GSH conjugates but in such cases GSSG levels do not increase. Arecoline itself is known to readily react with free thiols. So GSH conjugation of arecoline may deplete free thiols without actually producing

ROS. This mechanism seems to be unique to Arecoline as areca nut extract by itself can increase intracellular ROS (Chang et al 2001). 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. To understand these biological effects of autooxidation of pan masala components, we will have to first examine the complex interactions that take place between the individual components and chemical entities that make up the complex mixture of Pan masala.

1.9.2. Polyphenols in Pan masala constituents: Areca nut (around 11-17% polyphenols, which include tannins and flavonoids such as anthocyanidine and leucocyanidine) (Bhide 1976) and catechu (13-33%), two main constituents of Pan masala mixture contain significant amounts of plant polyphenols which have been known to exhibit opposing effects on genotoxicity depending upon concentration. So concentration of polyphenols in Pan masala is crucial in deciding the course of cellular response to Pan masala. Plant polyphenols have been known to work as both antioxidants and prooxidants. Some of the best-studied polyphenols (epigallocatechin gallate, catechin gallate) produce H2O2 in cultured cells (implicated in some of the antitumorigenic effects of polyphenols). The polyphenol molecules are autoxidized in the presence of dimolecular oxygen to produce superoxide and semiquinone radicals. The superoxide radical further oxidizes catechins to produce H2O2 and quinones (Mochizuki et al 2002 and Nair et al 1987). In a similar case, aqueous areca nut and catechu extracts were able to induce production of reactive oxygen species such as superoxide anion and hydrogen peroxide in the presence of lime in a cell free system at pH ≥ 9.5. However, it was maximum with tannin, the tannin fraction of areca nut extract (for O2

● ─

production) and catechol (H2O2

production) indicating a role for polyphenols in ROS production (Fig 7). Fig. 7 ROS production from polyphenols OH

O•

O O

OH

2H

pH ≥ 9.5 Autooxidation

OH•

+

H2O

+

O2

R

R

R

Haber-Weiss Reaction Quinone

Polyphenol

Semiquinone radical Superoxide dismutase

● ‾

Semiquinone radical + O2 H2O2

+

O‾

+

2+

Fe

O2

+

H2O2 (Reaction 6) 3+ 8) OH• + Fe +(Reaction H2O

+

H

Fenton’s Reaction ● ‾

O2

+

H2O2

+

H

+

Polyphenols can also act as ROS quenchers by

(Reaction 7)

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. R-OH ↔ R-O- + H+ R-OH + OH- ↔ R-O- + H2O

(Reaction 8) (Reaction 9)

1.10. Measurement of oxidative stress after Pan masala exposure: Pan masala is a popular chewing product in India and other countries. Its consumption has been linked with increased incidence of precancerous conditions. Pan masala and its components have been shown to possess genotoxic and carcinogenic properties. However, no strong carcinogens have been identified in Pan masala mixture to explain the link to oral cancer. Demonstration of ROS production after autooxidation of areca nut polyphenols (Nair et al 1987) was followed by similar observations for cultured cells exposed to aqueous Pan masala extracts (Bagachi et al 2002). However, very scant information is available for oxidative stress following Pan masala exposure in vivo. Oxidative stress/damage has been known to participate in various pathological processes including carcinogenesis. Hence, oxidative stress as a result of ROS formation, occurring after autooxidation of polyphenols present in Pan masala, appears as a likely candidate contributing to the biological activities of Pan masala. Hence, in this study, to investigate the role of oxidative stress in biological activities of Pan masala, the following objectives were decided1.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.