Scientific Officers: G.B. Maru, Ph.D. (Head), S.S. Pakhale, Ph.D., A.G. Ramchandani, Ph.D., A.N. Bagwe, Ph.D., M.B. Mahimkar, Ph.D.

Research Fellows: R. Krishnan, S.N. Erande, R.R. Patel, D. Anantharam, S.M. Pathare, R.K. Garg.

The role of environmental agents in human cancer causation and modulation forms the main theme of on-going research in the Tobacco Carcinogenesis group, wherein projects are broadly grouped under: (a) tobacco carcinogenesis – these encompass chemical analysis, assessing bioactivity of tobacco products, biomonitoring of habitues, identification of genetic alterations in oral tumours using molecular cytogenetic approach, and elucidating the role of DNA methylation in head & neck, and breast carcinogenesis, and (b) chemo-modulation of tobacco carcinogenesis. A sizeable effort is also being directed towards identification and delineation of the mechanism of chemopreventive action of agents from Indian food, and development of mechanism-based biomarkers.

Chemical analysis of tobacco / tobacco products and measurement of tobacco-related biomarkers of exposure

Tobacco and tobacco smoke are causative agents of a wide variety of cancers, cardiovascular and respiratory diseases. The objectives of this project include: (i) comparative chemical analysis of carcinogenic constituents / precursors in tobacco and mainstream/sidestream smoke (MS/SS) and assessment of their biological activity, and (ii) setting up and applying standardised methodologies for the measurement of biomarkers in experimental systems and tobacco exposed/unexposed subjects. Six aldehydes have been detected in µg quantities in MS and SS of all the smoking products. Significantly high levels of aldehydes are also contributed by non-tobacco constituent / product - namely tendu leaf and herbal bidi. Nine phenolic compounds - phenol, catechol and hydroquinone being predominant, have been detected and quantitated in MS and SS of Indian smoking products. Levels of total phenols in the SS of chutta and bidi are lower than that of Indian/US cigarette. Total PAH levels are higher in the SS of bidi than that of Indian cigarette. Future plans include determining tobacco-specific nitrosamine (TSNA) levels and examining the mutagenicity, cell transformation and carcinogenicity of MS/SS of Indian smoking products, as well as measuring tobacco exposure-related urinary metabolites of TSNA and haemoglobin adducts in exposed animals and in habitués.

Carcinogenic potential of gutkha and mechanism/s of its action (Indian Council of Medical Research / Indo-German)

This group had earlier reported mutagenic and carcinogenic activity of pan masala in experimental systems. The present project involves a comparative evaluation of the mutagenic, clastogenic and carcinogenic activity of gutkha in relation to that of pan masala, and possible mechanism/s of its action. Chemical analyses reveal that gutkha contains both nicotine (1.6–4.5 mg/gm) and arecoline (1.4-2.5 mg/gm) while only arecoline (1.3-2.5 mg/gm) is seen in pan masala. Significant levels of trace metals such as phosphorus, calcium, copper, magnesium, zinc and iron have been detected in these products. Mutagenicity testing using the Ames assay reveals that aqueous and ethanolic extracts of gutkha - brand P (APGE/EPGE) exhibit direct mutagenicity in TA98/TA100 and TA98 respectively. However the aqueous extract of gutkha - brand M and both aqueous and ethanolic extracts of pan masala are non-mutagenic. Genotoxicity testing reveals lower micronucleated cell frequency in APGE-treated human PBL cultures, while the proliferative potential of EPGE-treated cultures is affected with increasing dose. Based on the results of maximum tolerated dose estimation, long-term carcinogenicity of gutkha (brand P) mixed in diet at 5% and 2.5% level is presently being examined in 554 Swiss mice. Future plans include studying on the carcinogenic influence of lifetime exposure to gutkha and mechanism(s) of pan masala / gutkha-induced carcinogenesis.

DNA adducts as markers of exposure to environmental mutagens/carcinogens: Tobacco

In view of the exposure of millions of people worldwide to tobacco, it is necessary to monitor DNA damage in tissues/cells of tobacco-exposed individuals using highly sensitive methods. The objectives of this project are: (i) setting up and validating the 32P-post labelling analysis of DNA for the detection and measurement of tobacco-induced DNA modifications in experimental systems, and (ii) applying validated methods to measure DNA adducts in tissues/cells of tobacco users and habit-free subjects. Data reveal that topical application of equal amount of TPM / equal product-derived TPM from the MS/SS of Indian non-filter, filter and American filter cigarettes and bidis to the mouse skin induces PAH-DNA adducts. SS is relatively more genotoxic than MS, and the SS of bidi shows exceptionally high relative adduct labelling value as the TPM yield is 3 to 5 fold lower than SS and MS of other products. Topical application of curcumin(s) decreases PAH-DNA adducts derived from TPM of MS/SS in mouse skin while dietary administration of turmeric is ineffective. Topical application of MS and SS also increases the levels of the oxidized DNA base 8-OH-dG in mouse skin and several internal organs, although dose response is seen only in the lung. Turmeric pre-treatment results in a decrease in TPM-induced 8-OH-dG levels in some tissues. Future plans include measurement of tobacco-related DNA adducts in cells/tissues of exposed experimental animals and tobacco habitués/controls.

Polymorphism of CYP1A1, GSTM1 and XRCC1 genes and susceptibility to oral cancer (Department of Biotechnology)

Cytochrome P450s (CYP) and glutathione S-transferases (GST) are involved in metabolism of tobacco-derived carcinogens. Polymorphism at these gene loci can modulate the metabolism of tobacco-derived reactive intermediates and thus alter susceptibility to oral cancer. Allelic variation in XRCC1 gene, involved in the inefficient repair of DNA damage, can also increase the cancer risk. In this project, it is proposed to: (i) determine the frequency of polymorphism at GSTM1, CYP1A1 and XRCC1 gene loci in patients suffering from tongue cancer / pre-malignant lesions and in controls, (ii) analyse the influence of ‘at risk’ polymorphic genotype/s in oral cancer patients with different extent of tobacco exposure and (iii) determine the cancer risk conferred by the polymorphic allele/s singly and in combination with other ‘at risk’ genotypes. The frequency of GSTM1 null genotype is 52% (31/59) in patients while that in controls is 30% (14/46). The frequency of CYP1A1 (MspI) wild type allele is found to be 53% (8/15) while that of the heterozygous and homozygous mutant allele is 47% (7/15) and 0% respectively. At the XRCC1 (PvuII) locus, wild type allele frequency is 77% (34/44) while that of the heterozygous and homozygous mutant allele is 20% (9/44) and 2% (1/44) respectively. Future efforts will be directed towards increasing sample size in habit-free subjects with oral pre-cancer and tongue cancer. The link between oral cancer susceptibility and polymorphism at the CYP2E1 and NAT2 metabolic gene loci and other genes involved in DNA repair will also be investigated.

DNA methylation status of cell cycle regulatory genes in head and neck, and breast cancers

DNA methylation dysregulation of critical genes has been noted in several cancers, and methylation-induced anomalous expression or silencing of cell cycle regulatory genes has been suggested as the mechanism responsible for carcinogenesis. The present study examines the DNA methylation status of the cell cycle regulatory genes - Myf-3 and p21 (Waf-1), in tumour tissue versus adjoining normal tissue and PBL from patients suffering from juvenile nasopharyngeal angiofibroma (JNA) - a highly proliferative but benign head and neck tumour, and breast cancer to determine if methylation alteration of these genes correlates with proliferation or malignant conversion. Data show that the DNA methylation status of both these genes is unaltered in the 14 JNA cases studied to date, indicating that the highly proliferative status of these tumours is not associated with methylation alteration. On the other hand, Myf-3 is clearly hypermethylated in 40% (48/120) breast tumours. Bimodal methylation pattern is noted in p21 (Waf-1) gene with 18% (22/120) breast tumours showing hypermethylation and 31% (37/120) showing hypomethylation. Immunohistochemical staining for p21 is presently underway. The present findings thus point towards methylation dysregulation of p21 (Waf-1) and Myf-3 genes in breast cancer.

Mechanism(s) of turmeric / curcumin mediated chemoprevention (Indian Council of Medical Research)

Due to the chemopreventive properties of turmeric and its perceived human safety following centuries of use as food and medicine, turmeric and its active principle curcumin are today being considered for clinical development. Earlier studies from this laboratory had shown that turmeric and curcumin inhibit the formation of carcinogen-DNA adducts in vitro and in vivo, reduce the activity of carcinogen-induced isozymes of cytochrome P450 in tissues of animals, and decrease the multiplicity of tumours in experimental animals. Studies have now been initiated to understand the mechanism/s involved in turmeric and curcumin–mediated chemoprevention of experimentally induced tumours, and validation of surrogate endpoint biomarkers and parameters for drug effect measurement which may prove to be useful in monitoring chemoprevention trials.

Chemopreventive efficacy of black tea (Camelia sinensis) - a popular Indian beverage (National Tea Research Foundation)

Tea is one of the most popular beverages worldwide, and is generally consumed either as black tea or as green tea. During the manufacture of black tea, some of the characteristic green tea flavanols (shown to possess anti-oxidative and anti-carcinogenic activities) get reduced several fold while the characteristic black tea polyphenols - theaflavins and thearubigins are formed. Information on chemopreventive effects, if any, of these newly formed compounds is not available. This project proposes to evaluate the chemopreventive effect/s of black tea and its components on mutagen/carcinogen–induced alterations and tumorigenesis, and to elucidate the mechanism/s of their chemopreventive activity using in vitro and in vivo experimental models.

Biomarkers of oxidative stress as a measure of exposure to tobacco

Millions of Indians are habitual users of tobacco, which has been implicated as the causative agent for various cancers. Experimental studies indicate that exposure to tobacco or tobacco products results in oxidative stress, a process that may be associated with cancer development. Tobacco or tobacco products contain several constituents, including volatile aldehydes, which can cause oxidative stress and lipid peroxidation. Studies have been initiated to examine biomarkers or indicators of oxidative stress, namely (i) DNA adducts/damage, (ii) protein modifications and (iii) lipid hydroperoxides in cells/body fluids of tobacco users with or without pre-cancerous lesions as well as in control human subjects with no tobacco habits, employing highly sensitive methods.

Chemopreventive effects of polyphenolic components from Indian grapes (Indian Council of Medical Research)

Grapes are among the most widely consumed fruits in the world and are rich in polyphenols, which are known to exert chemopreventive effects in different experimental systems. No information is available on the bioactivity and mechanisms of action of crude polyphenolic fraction of grapes and their components on the carcinogenesis process. This project has therefore been initiated to evaluate the chemopreventive effect of polyphenolic components extracted from Indian grapes on carcinogen and/or co-carcinogen-induced alterations in different experimental systems.