S/RV Cri-ba, a hairless mouse strain sensitive to skin tumorigenesis by suboptimal doses of 7,12-dimethylbenz[a]anthracene, initiation-promotion and two stage promotion protocols.

Susceptibility of hairless inbred S/RV Cri-ba or Bare mice to skin tumor development with suboptimal doses of 7,12-dimethylbenz[a]anthracene (DMBA), DMBA-TPA two stage protocol and two stage promotion using 12-O-tetradecanoylphorbol-13-acetate (TPA) as sub-stage 1 promoter and mezerein (MEZ) or phorbol retinoate acetate (PRA) as substage 2 promoter was determined. A single application of 40 or 20 nmol DMBA induced 4-5 papillomas per mouse 40 weeks after initiation while no tumors appeared after similar treatment with 10 or 4 nmol DMBA. Dose response studies for DMBA initiation revealed that 10 nmol DMBA dose saturated the sites for initiation in the resting epidermis. In two stage promotion experiments, MEZ was found to be a potent stage 2 promoter, while PRA acted as a weak complete promoter.

Mutagenic activity in urine samples from female tobacco habitues.

Since high incidence of oral cancer in India is associated with smokeless tobacco usage, mutagenic exposure of subjects habituated to a pyrolysed tobacco product, masheri (M) and tobacco-containing betel quid (Q) was evaluated in the present study. Urinary cotinine was estimated to ascertain tobacco exposure and urine mutagenicity to Salmonella typhimurium TA98 and TA100 was used to assess mutagenic burden. Urinary cotinine levels were higher in MQ users than in M users. Urine mutagenicity was evident in control samples only upon treatment with S9, beta-glucuronidase or acidified nitrite. However, greater exposure of users to mutagens resulted in additional direct mutagenicity to TA100.

Effect of the active tumor promoter, 12-O-tetradecanoylphorbol-13-acetate on hair follicular growth and development of hair anlage tumors in the mouse skin: a comparison with human adnexal lesions.

Effect of the active tumor promoter, TPA, on the induction of hair follicular growth and possible neoplasia was studied histologically in normal nd precancerous mouse skin. Biweekly application of TPA resulted in nevoid proliferation of hair follicles, decreased the latent period of tumor induction, and increased the number of carcinomas of hair follicular origin in mice pretreated with MCA. The experimental tumor induction data was compared with human adnexal tumors of hair follicular origin. Similar types of animal and human lesions could be attributed to the promoter effect alone or to the dual influence of a carcinogen and promoter.

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.

12-0-Tetradecanoylphorbol-13-acetate-induced amplification of mesenchymal tumorigenesis in the mouse skin.

Skin tumors were induced in 6-week-old female Swiss albino mice by a single subcutaneous (SC) injection of 20-methylcholanthrene (MCA) in the right scapular region and the animals were then divided into four groups. Mice in group I did not receive further treatment. Six weeks after MCA injection, those in groups II and III received twice weekly applications of 0.1 ml acetone and 1.8 nmol 12-0-tetradecanoylphorbol-13-acetate (TPA) in 0.1 ml acetone, respectively, at the site of MCA injection until tumor development. Group IV animals were divided into four subsets and administered two, four, six, or eight TPA applications commencing 6 weeks after carcinogen injection. The effect of TPA pretreatment on MCA-induced tumorigenesis was studied in animals in group V. In mice treated with MCA alone, the most predominant mesenchymal tumor type is fibrosarcoma with induction of some rhabdomyosarcomas. Mixed mesenchymal tumors consisting of fibrosarcoma, rhabdomyosarcoma, or hibernoma were observed in only 12% of the animals. The number of animals bearing mixed mesenchymal tumors such as fibrosarcoma, rhabdomyosarcoma, hibernoma, and/or liposarcoma increased to 46% in mice receiving MCA + TPA until tumor development. Interestingly, liposarcomas were not found at all in animals treated with MCA alone. The data indicates that TPA application to precancerous mouse skin enhances mesenchymal tumorigenesis.

Differential effects of phorbol ester tumor promoters on 3-methylcholanthrene-induced epithelial and mesenchymal skin tumorigenesis.

The effects of TPA, PDD, PDB, PDA, or MEZ on epithelial and mesenchymal skin tumors induced by a s.c. injection of MCA were studied histologically. Group-I mice received only MCA. At 6 weeks after MCA injection, mice in groups II to VII received acetone, 1.8 nmol TPA, PDD, PDB, PDA, or 6.1 nmol MEZ respectively in 0.1 ml acetone twice weekly until tumor development. Alterations in skin tumor induction patterns were also studied in animals that had been exposed to TPA or acetone for 10 weeks prior to s.c. injection of MCA. Exposure of mouse skin to TPA before or after carcinogen administration increased 2- to 3.5-fold, the incidence of carcinoma and mixed tumors of epithelial and mesenchymal histogenesis. The average time of tumor induction decreased in mice treated with MCA + TPA and 100% of the test animals in the TPA + MCA group developed tumors. In contrast, TPA-related phorbol esters inhibited skin tumor development, particularly trichoepithelioma and fibrosarcoma and increased the average time of tumor induction.

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.

Effects of an aqueous extract of processed bidi tobacco on the growth of hamster tracheal epithelial cells.

Inhalation of tobacco dust is responsible for elevated genotoxicity and pulmonary ailments in workers engaged in processing tobacco for the manufacture of bidis, the Indian version of cigarettes. Tracheal tissue being the major site of interaction with tobacco dust, the effects of different concentrations of an aqueous extract of bidi tobacco (ATE) on the growth of a hamster tracheal epithelial cell line (HTE) were investigated. Colony forming efficiency assay revealed that ATE was cytotoxic only at the highest concentration of 5.0 mg/ml. In cultures treated with 1.25 mg/ml ATE, the cell doubling time and growth rate were similar to that of the controls, while a significant increase in cell doubling time (29.4+/-0.3 h vs 14.0+/-3.75 h, P<0.001) was observed at 2.5 mg/ml ATE concentration. Exposure of HTE cells to the non-toxic ATE concentration of 2.5 mg/ml was found to stimulate ornithine decarboxylase (ODC) activity, incorporation of [3H] methyl thymidine into DNA and increase in the S phase fraction was seen by flow cytometry. However, a 56% reduction in the growth rate of cultures treated with 2.5 mg/ml ATE was related to the prolongation of the traverse of cells through S phase. ATE-induced growth suppression was reversed when cultures were grown in ATE-free medium or upon repeated exposure to ATE. The findings suggest that increased tracheal cell proliferation induced by chronic inhalation of tobacco dust may contribute to the development of pulmonary disorders and possibly neoplasia in exposed individuals.

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.

Biological monitoring of bidi rollers with respect to genotoxic hazards of occupational tobacco exposure.

Smokeless tobacco habits are associated with a high incidence of oropharyngeal cancer in India. Hence, the biological effects of occupational exposure to smokeless tobacco used for making bidis (the Indian version of cigarettes) were studied in 2 groups of bidi rollers designated BR-K and BR-S and in control subjects with no tobacco habits. Specific tobacco exposure and the electrophilic burden were determined by estimating urinary cotinine and thioethers respectively. Urine mutagenicity was tested with the Ames assay using Salmonella typhimurium strains TA98 and TA100. While cotinine was not detected in control samples, the mean cotinine levels (mmole/mole creatinine) in the BR-K and BR-S groups were 0.79 +/- 0.30 and 0.09 +/- 0.03 respectively. Urinary thioether excretion (mmole/mole creatinine) was significantly elevated in the BR-S group 4.59 +/- 0.52; p less than 0.001) but it was lower in the BR-K group (0.54 +/- 0.08; p less than 0.001) compared to the control (1.83 +/- 0.34). Furthermore, beta-glucuronidase-treated samples from both groups of bidi rollers exhibited increased mutagenicity to TA98 compared to the control group; in addition, BR-S samples exhibited direct mutagenicity to TA98. The results show that occupational tobacco exposure modulates the glutathione conjugation pathway and increases the mutagenic burden of bidi rollers.

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 bidi tobacco increases chromosomal aberrations in tobacco processors.

In India, workers engaged in processing of tobacco for the manufacture of bidis (the indigenous substitute for cigarettes) are chronically exposed to tobacco flakes and dust via the cutaneous and nasopharyngeal routes. Hence, workers in a tobacco processing factory were monitored for chromosomal aberrations (CA) using peripheral blood lymphocytes as the test system. Cytogenetic analysis revealed a significant increase in deletion fragments and chromatid gaps in the exposed group. The frequency of aberrant metaphases and the proportion of individuals with CA were significantly higher in workers than in controls, indicating that occupational exposure to tobacco imposes considerable genotoxicity among tobacco processors.

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'.

Influence of smokeless tobacco exposure on detoxification status and chromosomal damage in male and female habitues.

In India, a large number of tobacco chewers and masheri users are chronically exposed to tobacco genotoxicants. Detoxification processes involving cellular glutathione (GSH) and glutathione S-transferases (GST) determine the outcome of exposure to environmental mutagens including those present in tobacco. Hence, in this study, GSH levels, GST activity, GSTM1 genotype and cytogenetic damage were determined using lymphocytes from 114 smokeless tobacco habitues and controls. The study groups comprised of male tobacco chewers, female masheri users, and age- and sex-matched controls. Irrespective of the tobacco habit, GSH levels and GST activity were higher in females than in males. In both the groups of habitues, GSH levels were similar to those in controls, while a significant reduction in GST activity was observed in tobacco chewers only. The frequency of cytogenetic alterations was significantly elevated in both the groups of habitues with respect to controls. However, break-type aberrations were more frequent in tobacco chewers while gaps were commonly observed in masheri users. Differences in the nature of chromosomal alterations in the two groups of habitues appeared to be related to variation in total tobacco exposure and gender-related differences in the efficacy of the GSH/GST detoxification system.

Influence of short-term topical exposure to phorbol ester tumor promoters on subcutaneously induced skin tumorigenesis in S/RVCri mice.

The effects of short-term exposure to phorbol ester tumor promoters on epithelial and mesenchymal skin tumor induction, studied histologically in female S/RVCri mice, rendered precancerous by a subcutaneous (sc) injection of 3-methylcholanthrene (MCA). At 6 weeks after MCA injection, mice in Groups I to VI received topical twice weekly applications of 0.1 ml acetone, 1.8 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol didecanoate (PDD), phorbol dibenzoate (PDB), phorbol diacetate (PDA) or 6.1 nmol mezerein (MEZ) in 0.1 ml acetone, respectively, for four weeks. Animals were sacrificed after the development of palpable tumors. Data from various phorbol ester treatment groups were compared with the acetone control. Of the promoters tested, TPA treatment increased the percentage of tumor bearers and the number of combinations and expression of diverse neoplasms in the mixed growths. TPA-related phorbol esters such as PDD, PDB or MEZ did not alter the percentage of tumor bearers although the histological distribution into pure epithelial or mesenchymal and mixed tumor bearers differed significantly from the control treated with the solvent alone. However, PDA significantly inhibited MCA-induced skin tumorigenesis and increased the average time of tumor induction. All the promoters, except TPA, decreased the percentage of mixed tumor bearers, the development of hibernoma and fibrosarcoma, while allowing a selective expression of rhabdomyosarcoma.

Ultrastructural analysis of epidermal hyperplasia induced by multiple 12-0-tetradecanoyl-phorbol-13-acetate (TPA) treatment of mouse skin.

Stationary epidermal hyperplasia induced by exposure of mouse skin to 1-6 TPA applications was analyzed by electron microscopy and found to be of two types. Intermingled orderly and irregularly stratified hyperplastic regions observed prominently after a single TPA application gave way, on multiple treatment, to epidermal hyperplasia populated by either cuboidal cells with expanded cytoplasm or by highly polar, narrow, tall and pleomorphic cells. Both cell types were poorly differentiated and displayed a paucity of intact desmosomal junctions, resulting in an incohesive tissue structure in which a number of phenotypic variants were expressed. The variants were markedly less mature than the adjacent cells and showed basal cell phenotype, acquisition of secretory activity or a disturbed mitotic process, resulting in the formation of binucleated cells. The observations suggest that the disturbed mitotic process, poor cellular differentiation and induction of metaplasia could be the mode by which an initiated cell may express its tumor phenotype and escape differentiation during the early stage of TPA promotion.

Correlation between tissue growth kinetics and modulation of mouse skin tumorigenesis by phorbol esters.

Previous studies on the influence of phorbol esters on mouse skin tumorigenesis have shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances development of malignant epithelial and mesenchymal skin tumors by a completely carcinogenic dose of 3-methylcholanthrene (MCA), while its congener phorbol-12, 13-diacetate (PDA) exerts an inhibitory effect. Differential effects of these two agents were analysed by histology, morphometry and cell kinetic techniques including autoradiography and estimation of labelled precursor incorporation into DNA by liquid scintillation counting. Epidermal hyperplasia induced on exposure of S/RV Cri mouse skin to a single or multiple TPA application after MCA injection was associated with a significant increase in the thickness of nucleated cell layers, stratum granulosum, number of suprabasal cells and dark basal cells. Enhancing effect of TPA on MCA-induced neoplastic development correlated well with an increase in mitotic activity, number of cells in S-phase and increased rate of DNA synthesis in the epidermis, dermis and subcutis as also mast cell number. In contrast, treatment of MCA-injected preneoplastic mouse skin with PDA resulted in epidermal hypoplasia and cellular damage evident as cytoplasmic vacuolation and nuclear pyknosis. Multiple PDA exposure also reduced the thickness, mitotic index and number of cells in S-phase in epidermis, dermis and subcutis. Thus, cellular toxicity and inability to recruit cells in DNA-synthetic phase may account for inhibition of progression of preneoplastic epithelial and mesenchymal cells into overt tumors by PDA.

Inversion of carcinogen-promoter sequence: effects on mouse skin tumorigenesis and cellular growth kinetics.

To stimulate conditions wherein humans might be exposed to tumor promoters prior to carcinogenic stimulus, female S/RV Cri mice were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 10 weeks followed by a sc injection of 3-methylcholanthrene (MCA). Six weeks after MCA administration, tissue alterations in different skin layers were analysed by histology, morphometry and autoradiography. Multiple application of TPA prior to MCA injection induced moderate to marked epidermal hyperplasia with an increase in the thickness of nucleated cell layers and stratum granulosum. As compared to control, number of basal and suprabasal cells per 7.5 mm of interfollicular epidermal (IFE) length was significantly higher in the skin of animals treated with TPA + MCA. The hyperplastic response was accompanied by a significant increase in epidermal mitotic activity, number of cells in DNA-synthetic phase in epidermis, dermis and subcutis and subepidermal mast cell population. Histological observations of induced tumors revealed a significant increase in the incidence of carcinomas and mixed neoplasms of epithelial and mesenchymal histogenesis. The findings suggest that stimulated cellular proliferation in different layers of mouse skin by TPA treatment prior to MCA injection may play a major role in enhanced expression of histogenetically distinct tumors.

Chemistry and toxicology of smokeless tobacco.

In most parts of the world, tobacco is used for smoking, whereas, in India, tobacco is used for smoking as well as in diverse smokeless forms. Absorption of toxic and carcinogenic chemicals in tobacco and other ingredients added to various products are causally associated with several non-communicable diseases including cancer, especially oral cancer, which is the leading cancer among men and the third most common cancer among women in India. This article highlights the toxicity, mutagenecity and carcinogenic effects of hazardous chemicals present in smokeless tobacco products. This endeavor was based on the extensive review of literature from various sources. The SLT products have influence on cellular metabolism, ability to cause DNA damage, and cancer in experimental animals. It is, therefore, essential to consider the collective role of chemical constituents of SLT products in the causation of adverse effect on human health.