Mutagenesis vol. 19 no. 5 © UK Environmental Mutagen Society 2004; all rights reserved.
Evaluation of DNA damage in mice topically exposed to total particulate matter from mainstream and sidestream smoke from cigarettes and bidis
Tobacco Carcinogenesis Group, Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai-410 208, India
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Abstract |
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The genotoxic potential of total particulate matter (TPM) from mainstream smoke (MS) and sidestream smoke (SS) of Indian smoking products, namely cigarettes and bidis, as well as a brand of US cigarettes, was studied by determining the levels of bulky aromatic DNA adducts in mouse tissues. TPM from MS or SS of various smoking products [equal weights (2.5 mg) or the amount derived from equal (0.25) cigarette/bidi] was applied topically to mouse skin once a day for four consecutive days and adduct levels were determined in DNA from skin and lung by 32P-post-labelling analysis. Relatively higher levels of bulky aromatic DNA adducts were noted in mouse skin treated with MS from a single Indian non-filter (INF) cigarette when compared with MS of a single bidi (with about half the product weight and one-quarter the tobacco compared with a cigarette), while comparable adduct levels were noted with SS from these two products. Considering the differences in the yields of constituents of tobacco smoke from the different products analyzed, the genotoxic potential of INF, Indian filter king (IFK) and American filter (AF) cigarettes as well as bidis was determined by topically applying an equal amount of TPM (rather than equal product-derived TPM). SS-derived TPM from all the products showed relatively higher levels of total polycyclic aromatic hydrocarbons and induced relatively higher levels of bulky aromatic DNA adducts than those derived from MS. The data indicate that TPM (MS + SS) from cigarettes appears to be more genotoxic than that from bidis and the contribution of tendu leaf (a non-tobacco bidi wrapper) to the generation of bulky aromatic DNA adducts appears to be significant, particularly in SS of bidis. Topical pretreatment with curcumin decreased the levels of TPM-derived adducts while pretreatment with dietary turmeric failed to show such protection.
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Introduction |
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Tobacco smoking has been implicated as a significant contributing factor in the aetiology of respiratory, cardiovascular and various neoplastic diseases. It is now well established that smoking is positively associated with the risk of certain cancers, such as lung, oral cavity, pharynx, urinary bladder, etc. (IARC, 1986
). The combustion of tobacco products leads to the formation of mainstream smoke (MS) and sidestream smoke (SS). MS is generated during puff drawing in the burning cone of the cigarette and SS arises mainly from passive burning of the cigarette/bidi between puffs and is the major contributor to environmental tobacco smoke (ETS). Chronic exposure to ETS pollutants has been identified as a factor in the induction of bronchitis, pneumonia and other diseases of the lower respiratory tract in children and also of lung cancer in non-smokers. Tobacco smoke, a complex mixture of more than 3800 chemicals, is known to induce a variety of bulky DNA adducts which are formed due to the metabolic activation of polycyclic aromatic hydrocarbons (PAHs) and aromatic amines present in the neutral fraction of cigarette tar. This fraction contains the greatest tumorigenic activity (Hoffmann and Wynder, 1986). In contrast to the voluminous data available on the harmful constituents of western tobacco products, there is very little information available on Indian tobacco products. These products are exported to several other countries and limited data have shown that yields of carcinogenic total particulate matter (TPM), nicotine, hydrogen cyanide, etc. are quite different among Indian smoking products and also when compared with those reported for western smoking products (Pakhale et al., 1997a,b; Pakhale and Maru, 1998; Dolas et al., 2001). In the present study the yields of total PAHs in MS and SS of Indian non-filter (INF), Indian filter king (IFK) and American filter (AF) cigarettes and bidis and their genotoxic potential in mouse skin have been compared.
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Materials and methods |
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Materials
Micrococcal nuclease (grade VI), calf spleen phosphodiesterase, RNase A, RNase T1, proteinase K, nuclease P1, curcumin and PEI–cellulose thin layer chromatogaraphy (TLC) plates were bought from Sigma Chemical Co. (St Louis, MO), T4 polynucleotide kinase (PNK) and carrier-free 32P-labelledorthophosphate were purchased from Amersham Pharmacia Biotech (Little Chalfont, UK) and the enzymes required for the synthesis of [
-32P]ATP were purchased from Boehringer Mannheim (Mannheim, Germany). The cigarettes and bidis were selected on the basis of their popularity/consumption and were purchased from the open market in Mumbai during 2000–2001. A brand of imported US cigarettes was purchased from the open market during the same period. All other products, turmeric and other reagents (AR grade) were purchased locally.
Animals
Inbred male Swiss bare mice (6–8 weeks old) were obtained from the animal colony of the Cancer Research Institute, Mumbai. They were randomly distributed into various groups, housed in cages (five per cage) and maintained under standard conditions of 22 ± 2°C, 45 ± 10% relative humidity and 12 h light/12 h dark cycle each day. All animals were fed powdered control/experimental diet and drinking water ad libitum.
Preparation of experimental diets
Turmeric rhizomes were purchased in a single lot from the local market in Mumbai, India, washed with water, dried and powdered in a grinding mill and stored at room temperature throughout the experiments. The curcumin content of turmeric was analyzed by preparative TLC (Roughley and Whiting, 1973). The curcumin(s) content (% weight of dry turmeric powder) was 5% and the proportions of the components curcumin, demethoxycurcumin and bis-demethoxycurcumin were 67, 28 and 5%, respectively. The quantities of turmeric powder required for preparation of a 1% turmeric diet were weighed and added to preweighed standard laboratory diet. Aldehyde-free ethanol (
5% concentration) was added to the turmeric diet and also to the standard laboratory diet to ensure its uniform distribution and was allowed to evaporate before the diets were used. Diets were prepared monthly and stored at 4°C. Food cups were replenished with fresh diet every day.
Collection of TPM from MS and SS of bidis and cigarettes
Prior to smoking, the cigarettes and bidis were conditioned for at least 24 h in a chamber at 60 ± 3% relative humidity and 22 ± 2°C. Owing to poor combustibility and to keep them from being extinguished, bidis need to be smoked with a minimum of two puffs per minute instead of the standard one puff per minute recommended for cigarettes. To achieve a meaningful comparison, the Indian cigarettes, bidis and a brand of US cigarettes were all smoked under the modified smoking conditions (2 puffs/min) while maintaining other international standards/parameters, i.e. 35 ml puff volume and 2 s puff duration. Non-filter cigarettes and bidis were smoked to a butt length of 23 mm and filter cigarettes were smoked down to the length of filter plus filter overwrap plus 3 mm in a humidity and temperature controlled laboratory. A single cigarette/bidi was smoked at a time and TPM in the MS and SS were collected separately on a Cambridge glass filter (3 cigarettes or bidis per filter) (Pakhale et al., 1997b). TPM (wet) was determined as the weight gain of the respective Cambridge filter pads. TPM from the filter was repeatedly extracted in acetone by sonication and dried under nitrogen (Lofroth and Lazaridis, 1986).
Determination of total PAHs in TPM
The TPM from the filters was repeatedly extracted in n-hexane by sonication. The PAH fraction in n-hexane was isolated by column chromatography on activated silica gel (Rothwell and Grant, 1974). Quantitative determination of the PAHs thus fractioned was carried out fluorimetrically using benzo[a]pyrene (B[a]P) as a standard at 
ex 387 and em 407.
Treatment schedule for measurement of DNA adducts induced by a single cigarette or bidi
The treatment schedule is presented in Table I. Forty Swiss bare mice were divided into 10 groups as follows: (1) vehicle control; (2) curcumin control; (3) TPM from MS of a cigarette; (4) TPM from SS of a cigarette; (5) curcumin + MS of a cigarette; (6) curcumin + SS of a cigarette; (7) MS of a bidi; (8) SS of a bidi; (9) curcumin + MS of a bidi; (10) curcumin + SS of a bidi.
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Curcumin (10 µmol) and MS/SS-derived TPM from a single cigarette or bidi was prepared in acetone. Acetone/curcumin (10 µmol), followed 10 min later by acetone/TPM from 0.25 cigarettes or bidis was topically applied to dorsal skin of mice once a day for 4 successive days. The vehicle control (1) and curcumin control (2) received acetone and curcumin, respectively, followed by acetone.
Groups 3 (MS of cigarette) and 4 (SS of cigarette) received one application per day of acetone followed by TPM derived from MS and SS of 0.25 cigarettes, respectively, for 4 successive days, while groups 5 (curcumin + MS of cigarette) and 6 (curcumin + SS of cigarette) received one application per day of curcumin followed by TPM derived from MS and SS of 0.25 cigarettes, respectively, for 4 successive days.
Similarly, groups 7 (MS of bidi) and 8 (SS of bidi) received one application per day of acetone followed by TPM derived from MS and SS of 0.25 bidis, respectively, for 4 successive days while groups 9 (curcumin + MS of bidi) and 10 (curcumin + SS of bidi) received one application per day of curcumin followed by TPM derived from MS and SS of 0.25 bidis, respectively, for 4 successive days.
Treatment schedule for measurement of DNA adducts induced by equal TPM
The treatment schedule is presented in Table II. Fifty-six Swiss bare mice were divided into 14 groups as follows: (1) vehicle control; (2) 1% turmeric control; (3) 10 mg TPM (2.5 mg x 4 applications) derived from MS of INF cigarette; (4) 10 mg TPM from SS of INF cigarette; (5) 10 mg TPM from MS of IFK cigarette; (6) 10 mg TPM from SS of IFK cigarette; (7) 10 mg TPM from MS of AF cigarette; (8) 10 mg TPM from SS of AF cigarette; (9) 10 mg TPM from MS of bidi; (10) 10 mg TPM from SS of bidi; (11) 1% turmeric + MS of INF cigarette; (12) 1% turmeric + SS of INF cigarette; (13) 1% turmeric + MS of bidi; (14) 1% turmeric + SS of bidi.
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Standard laboratory or 1% turmeric diet was fed to the respective groups for a period of 15 days and 2.5 mg TPM derived from MS and SS of cigarettes or bidis prepared in acetone was topically applied to dorsal skin of mice once a day for 4 successive days (12–15). The vehicle control (1) and 1% turmeric control (2) received standard laboratory and 1% turmeric diet, respectively, for 15 days and topical application of acetone vehicle once a day for 4 successive days (12–15).
Groups 3 (MS of INF cigarette), 4 (SS of INF cigarette), 5 (MS of IFK cigarette), 6 (SS of IFK cigarette), 7 (MS of AF cigarette), 8 (SS of AF cigarette), 9 (MS of bidi) and 10 (SS of bidi) received standard laboratory diet for 15 days. On days 12–15, 2.5 mg TPM from the respective smoking product was topically applied once per day.
Groups 11 (1% turmeric + MS of INF cigarette), 12 (1% turmeric + SS of INF cigarette), 13 (1% turmeric + MS of bidi) and 14 (1% turmeric + SS of bidi) were kept on the 1% turmeric diet for 15 days. On days 12–15, 2.5 mg TPM from the respective smoking product was topically applied once per day.
Isolation of lung and epidermal DNA from skin
Mice were killed 24 h after the last application and dorsal skin and the lungs were excised. The excised skin was placed in a 60°C water bath for 30 s and then immediately submerged in an ice/water bath. The epidermis was then removed from the dermis by gentle scraping. Epidermal DNA and lung DNA were then isolated, quantitated spectrophotometrically (Gupta, 1993) and analyzed for bulky aromatic DNA adducts.
32P-post-labelling assay
[-32P]ATP required for the post-labelling assay was synthesized in the laboratory by substrate level phosphorylation of ADP using carrier-free 32P-labelled orthophosphate by the procedure of Johnson and Walseth (1979). The specific activity of [-32P]ATP used in the present study was 3000 Ci/mmol based on the PNK-catalysed phosphorylation of a known amount of DNA nucleotides. DNA digestion, adduct enrichment and 32P-post-labelling of total and adducted nucleotides were carried out essentially as recommended (Phillips and Castegnaro, 1999). The amount of DNA used for adduct analysis was 12 µg/sample, while 4 ng DNA-derived nucleotides/sample was used for labelling normal (total) nucleotides. Adduct labelling was carried out using 100 µCi (3 µM) [-32P]ATP/sample, while 50 µCi [-32P]ATP was used for total nucleotide labelling.
Bulky aromatic DNA adducts were purified by 1-dimensional PEI–cellulose TLC in 1 M sodium phosphate, pH 6.0, overnight onto a Whatman 1 wick. Origin areas containing purified adducts were cut out and the donor cut-outs were attached to acceptor PEI–cellulose TLC plates (10 x 10 cm) with the aid of two button-type magnets for contact transfer of adducts and subsequent 2-dimensional separation [i.e. D3 (3.5 M lithium formate, 8.5 M urea, pH 3.5) and D4 (0.8 M lithium chloride, 0.5 M Tris–HCl, 8.5 M urea, pH 8.0)] as described by Lu et al. (1986).
Detection of adducts was done using a GS 525 Molecular Imager (Bio-Rad Laboratories) and quantitation of DNA adducts [diagonal radioactive zone (DRZ)] was carried out using Molecular Analyst software, version 1.4 (Bio-Rad Laboratories). PEI–cellulose TLC plates were exposed to a phosphor screen for 24 h. Counts from areas corresponding to adducts in control DNA plates were subtracted from the treated DNA plates and relative adduct labelling (RAL) values were calculated according to the formula RAL=(c.p.m.adducts/c.p.m.total nucleotides) x dilution factor.
Statistical analysis
The data were analyzed by Student's t-test (for comparison between two groups) or ANOVA (comparison among multiple groups) and P < 0.05 was considered to be significant. SPSS 10.0 statistical software was used for all analyses.
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Results |
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Determination of total PAHs in TPM from MS and SS of cigarettes and bidis
Quantitative yields of PAHs (ng/product, ng/puff, ng/mg product burnt or ng/mg wet TPM) in MS and SS of INF, IFK and AF cigarettes (1 versus 2 puffs/min) and bidis (2 puffs/min) determined employing B[a]P as a standard are presented in Figure 1. Yields of PAHs in Indian and US cigarettes generally increased in MS and decreased in SS with an increase in puff frequency. Similar changes in the deliveries of TPM and nicotine have been observed with an increase in puff frequency (Pakhale and Maru, 1998
). Comparison among all smoking products showed relatively higher yields of PAHs in TPM derived from SS than from MS, probably due to the relatively lower burning temperature and lower oxygen supply during SS generation. Bidis, despite having the lowest amount of tobacco among all products analysed (200 mg versus 850–1000 mg), showed only slightly lower yields of PAHs in TPM from MS and SS when compared with the respective yields of cigarettes (expressed per product or per puff) and yields were much higher in MS and SS of bidis than cigarettes when results were expressed per mg product burnt or mg wet TPM. The results suggest a significant contribution of PAHs from the tendu leaf wrapper of bidis and a probable role of poor combustibility of bidis in enhancing the levels of PAHs in SS.
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TPM derived bulky aromatic DNA adducts in vivo
To compare the DNA-damaging potential of TPM from MS and SS of a single INF cigarette and a bidi, levels of bulky aromatic DNA adducts were measured in tissue DNA of mice topically treated with TPM. Identical modified smoking conditions (2 puffs/min) were employed. Yields of TPM and physicochemical characteristics of the products compared are presented in Table III.
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Equal product-derived TPM-induced DNA adducts
Analyses of DNA adducts from skin of mice topically exposed to TPM from MS and SS of a single INF cigarette and bidi (0.25 product/day x 4) showed a DRZ, as reported (Randerath et al., 1986
, 1988). No DRZ was observed in DNA from vehicle-treated groups (Figure 2). TPM from MS derived from a single INF cigarette showed the highest RAL (4.66 ± 0.21), followed by MS of a bidi (2.91 ± 0.44), in DNA from skin epidermis. DNA adduct levels (RAL) from SS-treated skin of a single INF cigarette and a bidi were comparable (1.87 ± 0.91 and 2.06 ± 0.21, respectively) (Figure 3). While a DRZ could be detected in lungs of mice exposed to MS and SS (topical application on skin) from a single INF cigarette, no DRZ could be detected in lungs of mice similarly exposed to MS and SS from a single bidi. The adduct levels in MS-treated groups were significantly higher than those in SS-treated groups for both INF cigarette- and bidi-treated mouse skin. The adduct levels in lungs treated with MS and SS from an INF cigarette were 1.87 ± 0.19 and 0.45 ± 0.12, respectively (Figure 3).
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0.05; @,#significantly different from MS and SS respectively, P Bulky aromatic DNA adducts per product (MS + SS) were relatively higher in cigarettes than bidis, but a comparison of DNA adducts in MS and SS of cigarettes and bidis (per product) does not give a clear cut idea about comparative genotoxicity as yields of TPM and PAHs in MS and SS of cigarettes and bidis differ because of differences in type and amount of tobacco used in these products, type of wrapper, design of the product and other physicochemical parameters (see Table III). Analysis of levels of DNA adducts (RAL) when expressed per 10 mg TPM and 100 ng PAHs (Table IV) suggests that despite a relatively lesser amount of tobacco in bidis (
200 versus 800 mg in cigarette) and significantly lower yields of TPM in SS of bidis (7 versus 24 mg from a cigarette), adduct levels induced by SS of bidis were similar to those induced by SS of cigarettes. Analysis of levels of PAHs in MS and SS of cigarettes and bidis when expressed per mg TPM and mg nicotine (Table IV) shows that SS had relatively higher levels of PAHs than MS of the respective products and a significant portion of PAHs in SS from bidis appear to come from a non-tobacco source, i.e. the tendu leaf wrapper (which does not contain nicotine). A significant contribution of tendu leaf to the levels of a bidi smoke constituent has already been observed (Dolas et al., 2001). Since the comparison of DNA adducts in MS and SS of cigarettes and bidis (per product) was not between equals, further experiments were performed by topical application of equal amounts of TPM (rather than equal product-derived TPM) from various smoking products.
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Equal TPM-derived bulky aromatic DNA adducts
Topical application of equal TPM (2.5 mg/day x 4) derived from MS and SS of INF, IFK and AF cigarettes and bidis (Figure 4) induced bulky aromatic DNA adducts in mouse skin in the order: SS of AF cigarette > SS of bidi > SS of INF cigarette > MS of INF cigarette > MS of bidi
MS of AF cigarette > SS of IFK cigarette > MS of IFK cigarette. Among animals exposed to MS from various smoking products, INF showed the highest RAL (1.71 ± 0.35) and IFK the lowest (0.62 ± 0.08). In animals exposed to SS from various smoking products the highest RAL was observed with AF (2.78 ± 0.36) and the lowest with IFK (0.92 ± 0.11). Among equal TPM from MS and SS of Indian smoking products, SS of bidis and INF and MS of cigarettes showed similar levels of DNA adducts (Figure 4). DNA adducts per 10 mg TPM MS + SS of cigarettes were relatively higher than those induced by 10 mg TPM from MS + SS of bidis.
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Overall, these results suggest that TPM from SS smoke is relatively more genotoxic than that from MS smoke. Expression of higher levels of PAHs per mg nicotine (Table IV) in SS of bidis once again suggests a significant contribution of the bidi wrapper, i.e. tendu leaf.
Effect of topical pretreatment with curcumin on topical TPM-derived DNA adducts
Topical pretreatment of mice with curcumin(s) (10 µmol/day x 4) prior to topical application of TPM from MS and SS of a single cigarette or bidi (0.25 product/day x 4) (Figure 3) resulted in a significant decrease in the levels of bulky aromatic DNA adducts in skin and lungs of INF cigarette- as well as in skin of bidi-derived TPM-treated mice (Figure 3).
Reductions in adduct levels of 65 and 81% were observed in skin of mice pretreated with curcumin and subsequently treated with TPM from MS and SS from a single INF cigarette when compared with respective control groups. Similarly, mice pretreated with curcumin and then with TPM from MS and SS of a bidi showed decreases of 77 and 82% in adduct levels when compared with the respective control groups. A reduction in adduct levels of 58% was also observed in lungs (internal organ) of curcumin and cigarette MS-treated mice as compared with mice treated with cigarette MS alone. A substantial decrease in the levels of bulky aromatic DNA adducts was also observed in the lungs of mice topically treated with curcumin and cigarette SS, wherein no DRZ could be detected, as compared with the group treated with cigarette SS alone (Figure 3).
Effect of pretreatment with dietary turmeric on topical TPM-derived DNA adducts
Evaluation of the effect of dietary pretreatment of mice with turmeric (1%) on topical TPM (2.5 mg/day x 4) derived from MS and SS of cigarettes and bidis showed that dietary turmeric did not have a significant protective effect against the formation of bulky aromatic DNA adducts in skin of mice except in the group treated with turmeric + bidi-derived MS (Figure 4). This is probably due to the biologically effective doses of turmeric or turmeric-derived active components reaching the skin after dietary exposure being inadequate to counter the topical application of high doses of TPM. This may be a possibility as mice treated with dietary turmeric + MS of bidis showed a significant decrease in DNA adduct levels when compared with mice treated with bidi MS alone. The adduct level with MS of bidis was lowest, when compared with adduct levels in MS and SS of INF cigarettes and SS of bidis.
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Discussion |
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Smoking of tobacco is practiced world wide. Millions of people in India are exposed to tobacco smoke through various forms of smoking products. Bidi (representative of an eastern smoking product) smoking is more prevalent and widespread in India than cigarette smoking. These products are also exported to several other countries. Limited data on the comparative distribution of chemical constituents in tobacco smoke from Indian smoking products have shown that yields of tobacco smoke components in MS and SS are quite different between products and also from those reported for smoking products from the West (Pakhale and Maru, 1998
; Dolas et al., 2001). On the other hand, information on the relative genotoxicity of MS and SS of Indian smoking products is not available, while limited data on the relative genotoxicity of MS and SS are available for western cigarettes (Carmichael et al., 1993). Information on the relative DNA-damaging potential of tobacco smoke from these products is likely to help in evaluation and interpretation of experimental studies and may also help in estimating the relative contribution of genotoxic SS to the ETS from popular smoking products. To obtain meaningful comparisons the relative genotoxicities of equal product-derived or equal TPM from MS and SS of each of the smoking products were measured in a mouse skin model system by determining the levels of bulky aromatic DNA adducts, one of the early biomarkers of TPM-induced carcinogenesis and considered to be essential for tumour initiation. Since route of exposure, dose employed and target organ involved in this experimental study differ from those in smokers, it may not be possible to extrapolate these observations to interpret or predict smoking-related lung cancers in the East versus the West.
It may be noted that levels of total PAHs (whether expressed per product, per puff, per mg product burned or per mg wet TPM) were higher in SS than MS for all the products studied. Furthermore, in spite of the small amount of tobacco used in a bidi, emission levels of PAHs in SS of bidis were relatively higher, suggesting significant contribution of the bidi wrapper.
In spite of relatively higher levels of PAHs per product in SS than MS, bulky aromatic DNA adduct induction by INF cigarettes and bidis (per product) were higher for MS than SS. This is probably because of much higher amounts of TPM applied (obtained) in MS than in SS. Relatively higher levels of PAHs in SS than in MS in all the products studied is in agreement with earlier reports on western tobacco products wherein known carcinogenic PAHs have been shown to be formed in greater concentrations in SS than in MS (Grimmar et al., 1977; Hoffmann and Hecht, 1990). This is probably due to poorer efficiency of combustion during SS generation than during MS generation. TPM from MS and SS smoke of all smoking products produced characteristic DRZs (Figure 2), indicative of the reported formation of multiple DNA adducts in skin and lungs (Randerath et al., 1986, 1988; Carmichael et al., 1993; Randerath and Randerath, 1993). The DRZ probably contains a complex mixture of aromatic and/or hydrophobic adducts with a variety of structures and from a variety of chemical classes. It may also be noted that significant levels of bulky aromatic DNA adducts have been detected in the present study at much lower exposure levels (0.25 product-derived or 2.5 mg TPM/day x 4, i.e. one-third or one-seventh the exposure level) than in earlier studies (Randerath et al., 1986, 1988; Carmichael et al., 1993; Randerath and Randerath, 1993).
TPM from SS of cigarettes and bidis, on a per product or equal TPM basis, resulted in similar levels of DNA adducts while MS of bidis was relatively less genotoxic when compared with MS of cigarette on a per product or equal TPM basis. At equal TPM, SS appears to be relatively more genotoxic than MS for IFK and AF cigarettes and bidis, while MS and SS from INF show similar genotoxicity under the experimental conditions employed. It may be stated that even if the SS component of ETS is likely to be diluted considerably in air, the potentially harmful effects of passive smoking are evident at the molecular level. As with the levels of PAHs in SS of bidis, the levels of bulky aromatic DNA adducts in bidi smoke were remarkably higher and comparative expression of DNA adducts per mg nicotine once again indicated that the bidi wrapper (which does not contain nicotine) contributes significantly to the formation of TPM/PAHs and bulky aromatic DNA adducts. The contribution of tendu leaf to the MS of bidis is not striking, probably because of efficient burning and relatively more complete combustion of the tendu leaf during drawing (i.e. high cone temperature and higher availability of oxygen).
Overall, at equal TPM higher levels of carcinogens in SS from different smoking products were translated into greater adduct-forming potential in mouse skin, as reported earlier (Carmichael et al., 1993). When bulky aromatic DNA adducts induced by MS + SS of a product (INF cigarette or bidi) and equal TPM from MS + SS of INF cigarettes and bidis are compared, cigarette-derived TPM appears to be relatively more genotoxic than that derived from bidis. When levels of bulky aromatic DNA adducts were compared between equal TPM-induced versus equal product-induced, dose-related increases in adducts were noted with TPM from MS of both cigarettes and bidis, while such increases were not evident in TPM from SS of both cigarettes and bidis. The exact reasons for these differences are not known, although absorption of SS-derived compounds may be higher due to a relatively alkaline pH when compared with that of MS. In addition, the natures and types of PAHs and differences in the levels of moisture in TPM from MS and SS of various products may also have contributed to such differences. Topical pretreatment of mouse skin with curcumin(s) decreased the levels of bulky aromatic DNA adducts induced by a TPM-derived complex mixture of chemicals in skin and lungs. This observation is in agreement with reports demonstrating curcumin-mediated inhibition of single PAH (B[a]P or dimethylbenz[a]anthracene)-derived DNA adduct formation in vitro and in vivo (Lahiri et al., 1991; Huang et al., 1992; Mukundan et al., 1993; Deshpande and Maru, 1995; Singletry et al., 1996; Thapliyal et al., 2002). Dietary turmeric has also been shown to decrease the formation of oral B[a]P-induced DNA adducts in forestomach, liver and lungs of mice (Thapliyal et al., 2002), while in the present study dietary administration of turmeric (1%) prior to and during exposure to TPM did not cause a decrease in the levels of bulky aromatic DNA adducts in the skin. This is probably due to inadequate doses of the active principles of turmeric reaching the skin and an inability to protect against topical treatment with high doses of TPM. Low circulating levels of curcumins have been reported after dietary exposure of rats to turmeric (Ravindranath and Chandrasekhara, 1980).
In spite of employing a standardized methodology for collection of TPM from bidis and cigarettes, possible variations in collection of TPM from bidis cannot be ruled out. This is due to bidis being a product of rural origin, hand rolled by village artisans spread almost all over India. There is no standardization of the product other than shape. Manufactured bidis vary widely in weight, length and filling density, all of which profoundly affect the smoke characteristics. In addition, the smoking behaviour of bidi smokers may also vary. In Mumbai bidi is smoked at an average of 5 puffs/min, while our data are based on machine smoke generated at 2 puffs/min.
Overall, this report demonstrates differences in the relative genotoxicity of MS and SS from Indian smoking products, especially bidis. Further studies are needed with different brands of bidi and the types and concentrations of PAHs in MS and SS of cigarettes and bidis also need to be determined. Experiments may also need to be carried out after single topical application of TPM (rather than multiple applications) to fully understand and evaluate the relative genotoxicity of MS and SS.
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Acknowledgments |
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The authors thank Dr A.N.Bagwe for useful discussion, Mr R.Salgaonkar for the preparation of smoke condensates, and Mr P.Phase for technical help and the Indian Council of Medical Research (ICMR) for partial financial support of the project.
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Notes |
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1 To whom correspondence should be addressed. Tel: +91 22 27405022; Fax: +91 22 27412894; Email: gmaru{at}actrec.res.in
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References |
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Received on May 13, 2004; accepted on August 16, 2004.
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