Introduction: The use of smokeless tobacco (ST) is increasing world wide. The use of ‘gutkha’ (a form of ST) has been classified as carcinogen to humans and may be associated with oral diseases. So far very few systematic investigations have been done to reveal the molecular mechanisms involved in different types of ST-related health effects. Apart from oral cancer, the molecular mechanisms involved in other ST-related health effects are unknown. Therefore, the effects of chronic use of aqueous extract of ‘gutkha’ on cytochrome p450 (cyp) mediated genotoxic damage in liver, lung, and kidney of male Wistar rats were studied.

Materials and Methods: The experiments were performed on pathogen-free young male Wistar rats weighing 100-120 g after the study was cleared by the Institute’s Animal Ethics Committee. The aqueous extract of gutkha was lyophilized and orally administered to animals (n=7, in each group) at a low-dose (96 mg/kg b.wt/d) for 2 and 32 weeks, and at a high-dose (960 mg/kg b.wt/d) for 2 weeks. The immunohistochemical expression of Phase-I (cyp1A1, 1A2, 2E1) and Phase-II (GST-μ) enzymes; gene expression of p53, NF-κB, Bcl-2 and Bax by RT-PCR; myeloperoxidase activity, protein carbonylation, caspase-3 activity, DNA fragmentation and blood micronuclei formation (assay for chromosomal damage) were measured in lung, liver and kidney of rats.

Results: Our study shows that chronic use of aqueous extract of ST induces the differential immunohistochemical expression of xenobiotic phase-I and phase-II enzymes in various organs like lung, liver and kidney of rats. Immunohistochemical studies suggest that the expression of cyp2E1 in the chronic group was significantly higher as compared to cyp 1A1 and 1A2 in the lungs and kidney. There was significantly enhanced (p<0.05) activity of myeloperoxidase, protein carbonyl content, caspase-3 activity and DNA fragmentation in the above organs. The whole blood analysis in the chronic group confirmed the formation of micronuclei. Chronic use of ST significantly enhanced the gene expression of Bax, p53 and NF-κB and decreased the expression of Bcl-2 in a differential manner in various organs. However, the changes observed were greater in lungs and kidneys as compared to liver.

Conclusions: Chronic use of ST-induced expression of phase-I and phase-II enzymes mediates differential genotoxic damage in various organs of rats by modulating the apoptotic-antiapoptotic signaling pathways. The differential cellular damage by ST may be due to variable sensitivities and functions carried out by these organs. These changes might create a microenvironment favorable for ST-induced pathogenesis and initiate the process of carcinogenesis in different organs. Extensive research is further needed to decipher the molecular mechanisms involved in the ST-induced pathogenesis.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA