Skin-tumour-promoting activity of processed bidi tobacco in hairless S/RV Cri-ba mice.

Workers engaged in processing tobacco for the manufacture of bidis, the most popular smoking devices in India, are exposed to tobacco dust, volatile components and flakes via nasopharyngeal and cutaneous routes. In order to evaluate the risk of occupational tobacco exposure, the complete carcinogenic action of an aqueous extract of bidi tobacco (ATE), its ability to initiate and promote skin papillomas and to convert these to carcinomas, was tested in hairless S/RV Cri-ba mice using the skin tumorigenesis protocol. Epidermal cell kinetics and tissue alterations were recorded after a single or multiple applications of ATE to 7,12-dimethylbenz[a]-anthracene(DMBA)- initiated mouse skin. While ATE did not exhibit complete carcinogenic, initiating or progressor activity, it effectively promoted skin papilloma formation in DMBA-initiated mice. An increase in papilloma yield per mouse above the control was noted only after 30 weeks of promotion, and at week 40 of promotion with 5 mg and 50 mg ATE it was significantly higher than that in the control mice (9.69 +/- 1.30 and 11.73 +/- 1.38 compared to 4.70 +/- 1.01; P < 0.01). Mild epidermal hyperplasia, increase in mitotic activity and dermal thickness induced by a single application of ATE persisted upon multiple treatment and correlated well with its tumour-promoting activity. The findings indicate that occupational exposure to bidi tobacco may pose a cancer risk among workers in the bidi industry.

Evidence that DNA methylation imbalance is not involved in the development of malignant mesothelioma.

Methylation dysregulation has been a consistent finding in various malignancies, particularly those where the pathogenetic mechanisms are unclear. In order to test the hypothesis that methylation imbalance may not be a feature of cancers where the aetiologic agent or process is known, we studied the methylation status of the myogenic genes Myf-3 and Myf-4 by Southern blotting in malignant mesothelioma, a cancer strongly associated with asbestos exposure. DNA samples obtained from controls and mesothelioma patients did not exhibit hypermethylation of Myf-3 and hypomethylation of Myf-4, as noted in malignant lymphomas. The methylation status of Myf-3 and Myf-4 in malignant mesothelioma was similar to that of non-malignant cells indicating that dysregulation of the DNA methylating machinery may not be involved in mesothelioma development. The present findings do not support the view that methylation imbalance is a consequence of neoplastic transformation, but indicate that it may be one of the early molecular events involved in the genesis of some cancers.

Biological monitoring of bidi industry workers occupationally exposed to tobacco.

Ambient and biological monitoring was undertaken among tobacco processors who are chronically exposed to tobacco particulates via nasopharyngeal and cutaneous routes. Ambient monitoring revealed that the inspirable dust concentration was 150-fold higher in the tobacco factory than in the control environment, and was associated with chronic bronchitis in workers. Increased systemic exposure to tobacco constituents was evident from the high levels of cotinine, thioethers, promutagens and direct acting mutagens in workers' urine. The mean glutathione level and glutathione S-transferase activity were significantly lower in the peripheral blood lymphocytes of workers; however, the frequency of the GSTM1 null allele was similar to that in controls. A significant increase in chromosomal damage was noted in target and non-target cells of tobacco processors. In view of the association between tobacco use and several non-communicable diseases, the findings of the present study indicate an urgent need to minimize tobacco exposure among the processors.

Mutagenicity of processed bidi tobacco: possible relevance to bidi industry workers.

The genotoxic potential of bidi tobacco was evaluated by mutagenicity testing of aqueous, aqueous: ethanolic, ethanolic and chloroform extracts of processed tobacco used in the manufacture of 'bidis', indigenous forms of cigarettes smoked in India. The Salmonella/mammalian microsome test (Ames assay) was used to detect mutagenicity in tester strains TA98, TA100 and TA102. The extracts were tested in the absence and presence of metabolic activation using liver S9 from rat and hamster, and following in vitro nitrosation with sodium nitrite at acidic pH. All the extracts were non-mutagenic in the absence of nitrosation. The nitrosated aqueous extract was mutagenic in strains TA98 and TA100. While weak mutagenicity was elicited by the nitrosated aqueous: ethanolic extract in TA100, the nitrosated ethanolic extract induced a 3-fold increase in the number of revertants in the same strain. Moreover both these extracts elicited a strong mutagenic response in TA102, while the chloroform extract was non-mutagenic even after nitrite treatment. The present study indicates that workers employed in the bidi industry are exposed to potentially mutagenic and genotoxic chemicals in the course of their occupation.

Occupational exposure to tobacco and resultant genotoxicity in bidi industry workers.

In India, over 3 million workers employed in the bidi industry receive massive, chronic exposure to unburnt tobacco, mainly by the cutaneous and nasopharyngeal routes. While the hazards of habitual tobacco usage are well established, very little information is available about the effects of occupational tobacco exposure. In the present study, tobacco processing plant workers (TPPW) and bidi rollers (BR) with or without tobacco habits were monitored for occupation-related exposure to tobacco and resultant genotoxicity. Salivary cotinine levels were determined as an index of tobacco exposure and micronucleated buccal epithelial cell (MNC) frequency was recorded as a genotoxic endpoint. Occupational tobacco exposure led to the detection of cotinine in the saliva of 19% of BR and 100% of TPPW with no tobacco habit (NH). The greater degree of exposure in TPPW was evident from the significantly higher mean salivary cotinine level in TPPW-NH as compared to BR-NH (2.86 +/- 0.48 vs. 0.84 +/- 0.26 micrograms/ml; p < 0.01). The effect of occupational exposure was also evident in TPPW and BR with the masheri habit. A moderate but statistically significant increase in MNC frequency was observed in habit-free as well as masheri-habituated TPPW and BR as compared with the respective controls. The findings provide preliminary evidence for the clastogenic effects of occupational tobacco exposure.

Evaluation of the mutagenicity of 'pan masala', a chewing substitute widely used in India.

Mutagenicity of polar and non-polar extracts of a popular brand of 'pan masala' was examined using the Salmonella/mammalian microsome test (Ames assay) and 2 tester strains of Salmonella typhimurium, TA98 and TA100. These extracts were also subjected to pretreatment with sodium nitrite at acidic pH, to simulate conditions for endogenous nitrosation. The aqueous, aqueous:ethanolic and chloroform extracts as well as their nitrosated mixtures were non-mutagenic in the Ames assay, in the presence and absence of metabolic activation. Only the ethanolic extract elicited a weak mutagenic response in strain TA98 without metabolic activation demonstrating the presence of direct-acting frameshift mutagens in 'pan masala'.

Occupational exposure to unburnt bidi tobacco elevates mutagenic burden among tobacco processors.

The nature of mutagenic burden due to occupational exposure to tobacco flakes and dust was determined among 20 female tobacco processors (TP) and 20 matched controls (C) by testing urinary mutagenicity in the Ames assay. In addition, urinary cotinine was estimated as a marker of tobacco absorption. Workers and controls were sub-divided into those with no tobacco habit (NH) and those habituated to the use of masheri (a pyrolysed form of tobacco) as a dentifrice (MH). Cotinine was not detected in samples from C-NH while the mean urinary cotinine levels in TP-NH and TP-MH were significantly higher than that in C-MH (3.46 +/- 0.95 and 3.57 +/- 0.46 versus 1.80 +/- 0.58 mM/M creatinine; P < 0.02). The majority of the urine samples from C-NH were non-mutagenic in the presence or absence of rat liver S9 while those from C-H were mutagenic to TA98 and TA102 strains upon metabolic activation. On the other hand, direct mutagenicity to TA98, TA100 and TA102 strains respectively was noted in 6/10, 5/10 and 8/10 samples from TP-NH and 7/10, 4/10 and 3/10 samples from TP-M. Generally, beta-glucuronidase treatment reduced or abolished the mutagenic potential of workers' urine samples indicating that glucuronide conjugates may have partially contributed to direct mutagenicity. Experiments using scavengers of reactive oxygen species revealed that direct mutagenicity in TA102 strain was mediated mainly via hydroxyl radicals. The results clearly demonstrate that tobacco processors are exposed to a wide spectrum of mutagens that cause frame-shift, base pair substitution and oxidative damage.

Mutagenic potential of Indian tobacco products.

The mutagenic potential of aqueous extracts of masheri (ME), chewing tobacco alone (CTE) and a mixture of chewing tobacco plus lime (CTLE) was tested using the Ames assay. ME exhibited mutagenicity in Salmonella typhimurium TA98 upon metabolic activation with aroclor-1254-induced rat liver S9, while nitrosation rendered it mutagenic in TA100 and TA102. CTE exhibited borderline mutagenicity in the absence or presence of S9 in TA98 and TA100 and after nitrosation in TA102, while nitrosation led to doubling of TA98 and TA100 revertants. In contrast, CTLE exhibited direct mutagenicity in TA98, TA100 and TA102, was mutagenic to TA98 upon S9 addition and induced mutagenic responses in all three tester strains after nitrosation. Experiments using scavengers of reactive oxygen species (ROS) suggested that CTLE-induced oxidative damage in TA102 was mediated by a variety of ROS. The high mutagenic potency of CTLE vis à vis that of CTE may be attributed to changes in the pH leading to differences in the amount and nature of compounds extracted from tobacco. Thus, exposure to a wide spectrum of tobacco-derived mutagens and promutagens may play a critical role in the development of oral cancer among users of tobacco plus lime.

A novel MspI PCR-RFLP in the human cytosine 5-methyltransferase gene: lack of relevance for malignant lymphoproliferative disease and breast cancer.

Two alternate allelic forms of human cytosine 5-methyltransferase, 5-MT I and 5-MT II, which differ by the absence or presence of an intronic MspI recognition sequence, have been recognised. The polymorphic region was localised using a series of subprobes prepared upon MspI digestion of the 2.5-kb cDNA probe (hmt-2.5). A PCR-based method was then developed for rapid 5-MT genotyping. The gene and phenotype frequencies of 5-MT I and 5-MT II were not significantly different in genomic DNA samples from a series of non-Hodgkin's lymphomas and breast cancer cases compared with DNA from normal subjects. Allelism of 5-MT allows new approaches to the assessment of variation in gene copy number of 5-MT in different types of neoplasia.