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

Mutation Research, 261 (1991) 93-99 © 1991 Elsevier Science Publishers B.V. All rights reserved 0165-1218/91/$03.50 ADONIS 016512189100136V

93

MUTGEN 01698

Mutagenicity of processed bidi tobacco: possible relevance to bidi industry workers A p a r n a N. B a g w e a n d R a j a n i A. B h i s e y Carcinogenesis Division, Cancer Research Institute, Tata Memorial Centre, Parel, Bombay - 400 012 (India)

(Received 16 November 1990) (Revision received 26 February 1991) (Accepted 5 April 1991)

Keywords: Bidi tobacco, processed; Polar and non-polar extracts; Mutagenicity; Ames assay

Summary 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 $9 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.

A large number of human cancers are known to arise as a direct consequence of environmental exposure to mutagenic and carcinogenic agents, mainly through diet, habits and occupation (Tomatis et al., 1989). Recent studies have shown that occupational exposure to complex chemicals in the rubber, leather, chemical and dyestuffs

Correspondence: Dr. (Mrs.) R.A. Bhisey, Carcinogenesis Division, Cancer Research Institute, Tata Memorial Centre, Parel, Bombay - 400 012 (India).

industries in particular, poses a major carcinogenic risk (Preussmann, 1984). In India, about a third of all new cancer cases are related to tobacco habits (Sanghvi, 1989). Tobacco chewing and smoking habits have been closely associated with increased risks for oropharyngeal and lung cancers respectively (Peto and Zaridze, 1986). The vast majority of Indian smokers use 'bidis', which are indigenous, cheaper substitutes for cigarettes. The bidi manufacturing industry is a major industry employing a task force of ~ 3 million people (Jaisani, 1989). Processing of to-

94 bacco (Nicotiana tabacum) for the manufacture of bidis involves: harvesting, sun-drying, separation of leaves from the stalk, shredding of leaves, sorting, sieving, grading and admixture of fixed proportions of leaves from different cultivars and grades with separated stems and ribs, to achieve the desired composition and combustibility. Inside a tobacco processing plant, pounding, separation and sieving of tobacco is done manually. As a result, workers are exposed to a work environment heavily laden with coarse particles as well as fine, respirable tobacco dust. Unlike habitu6s who receive systemic exposure when tobacco is chewed, processing plant workers receive chronic exposure to tobacco mainly by the cutaneous and nasopharyngeal routes. In spite of such heavy exposure, no information exists regarding the mutagenic potential of processed bidi tobacco. Therefore in the present study bidi tobacco was subjected to polar and non-polar solvent extraction and tested in the Ames assay (Ames et al., 1975), in the presence and absence of metabolic activation. In addition, extracts were pretreated with sodium nitrite at acidic pH to generate conditions for in vitro nitrosation, and then tested in the Ames assay. Material and methods

Chemicals Histidine, biotin, glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate, reduced nicotinamide adenine dinucleotide phosphate (NADPH), dimethyl sulfoxide (DMSO), 4-nitroo-phenylene diamine (NPD), mitomycin C (MMC) and benzo[a]pyrene ( B ( a ) P ) w e r e obtained from Sigma (U.S.A.). Agar and nutrient broth were purchased from Difco (U.S.A.). All other chemicals used were analytical-grade reagents. Bidi tobacco Sun-dried uncured tobacco used for the manufacture of bidis was obtained in a single lot from a tobacco processing plant in South India. Finely powdered bidi tobacco was subjected to the following extraction procedures, sterilized by passing through a 0.20-/zm Millipore filter and stored at - 20 ° C prior to testing.

Aqueous extraction. The procedure of Guttenplan (1987a) was followed, wherein 10 mM phosphate buffer at pH 7 (10 vols./g) was added to the tobacco and extraction carried out at room temperature (30 o C) for 24 h. The extract was centrifuged at 18,000 rpm for 15 min and the supernatant lyophilized to achieve a 4-fold concentration. Hot aqueous : ethanolic extraction. Eight volumes of glass-distilled water : ethanol (1 : 1, v / v ) were added per gram of tobacco and the mixture refluxed in a constant-temperature water bath for 8 h. The extract was filtered to remove coarse particulate matter, the alcoholic component recovered by distillation and extracted material was left behind in the aqueous phase. Cold ethanolic extraction. Redistilled ethanol (2 vols./g) was added to the tobacco and extraction was done on an automatic shaker at 10 ° C for 2 h (Shah et al., 1985). The extract was stored overnight at - 2 0 ° C, filtered, dried on a rotary evaporator and then solubilized in glass-distilled water. Hot chloroform extraction. Eight volumes of chloroform per gram of tobacco were used for extraction in a Soxhlet apparatus. The extract was then subjected to filtration and solvent recovery, whereby a viscous, concentrated extract was obtained. Dilutions of this extract in DMSO were tested for mutagenicity. In vitro nitrosation. The procedure of Whong et al. (1984) was used, except that instead of using equal volumes of a 15 m g / m l aqueous solution of sodium nitrite and the extract, sodium nitrite concentration was kept constant at 300 / z g / p l a t e , since concentrations above 400 /zg/plate were mutagenic to TA100 (data not shown). Briefly, each extract and the sodium nitrite solution were dispensed in 2 separate tubes and the p H of each was adjusted to 3 using 1 N HC1. Different volumes of the extract were incubated with 3 0 0 / z g nitrite at 37 ° C for 2 h on a shaker, and the reaction mixture was tested in the Ames assay. Two independent experiments were carried out using freshly nitrosated extract.

95

Bacterial strains T h e histidine-deficient tester strains of Salmonella typhimurium, TA98, TA100 and TA102, were kindly provided by Prof. Bruce N. Ames (University of California-Berkeley, U.S.A.). Master plates were prepared every month from frozen permanents maintained at - 80 ° C. Genotype testing and responses to standard mutagens were confirmed at regular intervals (Maron and Ames, 1983). Metabolic activation system Aroclor 1254-induced male Sprague-Dawley rats were used to obtain the 9000 x g liver microsomal fraction ($9). Protein content of $9 was determined by the method of Lowry et al. (1951). High (10%) $9 mix prepared as per the procedure of Maron and Ames (1983) was used in all experiments requiring metabolic activation. Mutagenicity assay The preincubation protocol of Yahagi et al. (1985) was followed. The extracts (or reaction mixes) were incubated with 100/zl of $9 mix (or buffer) and 100 /xl of freshly grown bacterial culture (1-2 x 108 bacteria) for 20 min at 37 ° C. 2 ml of histidine-poor soft agar at 45 °C was added to the tubes and the mixture poured onto minimal-glucose agar plates which were incubated at 37 ° C for 48 h. Reversion from histidine auxotrophy to prototrophy was assessed by scoring revertant colonies. Using this protocol, reversion induced by NPD ( 2 0 / z g / p l a t e ) was 2130 _+ 66 and 450_+ 35 revertants per plate in TA98 and TA100 respectively, while MMC (0.5 /~g/plate) induced 2012 _+ 26 revertants/plate in TA102. In the presence of metabolic activation system, B(a)P (0.2 /zg/plate) induced 152 + 11, 402 _+ 41 and 551 + 13 revertants/plate in strains TA98, TA100 and TA102 respectively. Doses of all extracts were based on the dry weight of the original tobacco. A wide range of extract concentrations was initially tested to obtain a toxic dose, and further experiments were carried out at doses below toxicity. Sparse background lawn was considered to be an indicator of toxicity. Results represent the means of 2 independent experiments using duplicate plates. A 2-fold increase over the spontaneous reversion

rate along with a dose-related response was considered mutagenic.

Modified protocol for metabolic activation The procedure of Guttenplan (1987a) was followed. Syrian golden hamsters induced with Aroclor 1254 were used to obtain liver microsomal fraction (Maron and Ames, 1983). To different volumes of the aqueous bidi tobacco extract (ATE), 50 ~1 of a 2-fold concentrated culture of strain TA100, 120 ~I of hamster liver $9 and 380 ~1 of NADPH-generating system (Lee and Guttenplan, 1981) were added. The pH of the incubation mix was kept weakly acidic (pH 5.5), in order to maximize the mutagenicity of nitrosamines. The tubes (triplicates) were incubated at 37 °C for 45 min, following which 40/xl of aqueous N A D P H solution (50 m g / m l ) were added and incubation continued for an additional 45 min at 37 ° C. Histidine-poor soft agar (2 ml) was then added to each tube and the contents poured onto minimal-glucose agar plates. Revertant colonies were counted after incubation at 37 ° C for 48 h. Results

All 4 extracts were non-mutagenic in TA98, TA100 and TA102 both in the presence and in the absence of metabolic activation (data not shown). However, pretreatment of the aqueous, aqueous:ethanolic and ethanolic extracts with sodium nitrite at pH 3 rendered them mutagenic. The chloroform extract of bidi tobacco was nonmutagenic even after nitrosation (data not shown). The findings of mutagenicity testing of the nit r o s a t e d aqueous, a q u e o u s : ethanolic and ethanolic extracts are given below.

Nitrosated aqueous bidi tobacco extract (N-ATE) N-ATE was tested in the range 0.5-100 mg equivalent. In strain TA98, N-ATE was mutagenic in the absence of $9, showing a clear dosedependent increase in revertant numbers from 12.5 to 62.5 mg equivalent doses (Table 1). Maximum mutagenicity with a 5-fold increase (103 _+ 14) over spontaneous revertants was seen at 62.5 mg. Addition of $9 rendered the sample nonmutagenic. Table 1 also shows that in strain

96 TABLE 1 M U T A G E N I C I T Y O F N I T R O S A T E D A Q U E O U S E X T R A C T OF BIDI TOBACCO Dose

TA98

TA100

TA102

(mg)

- $9

+ $9

- $9

+ $9

- $9

+ $9

0a 12.5 25 37.5 50 62.5 75

29+2 44+4 66+2 68 _+4 83 + 3 103-+4 * 67+5

36+2 31+2 45+3 45 + 2 41 -+ 5 50+6 46_+6

138+7 257+8 299_+9 * 129 + 5 TR TR TR

134+ 6 175+ 4 2445:11 246 + 14 255 -+ 8 359+ 6 360+ 4 "

315+15 325+18 329+15 309 _+15 336 + 17 311-+ 9 298+ 8

363+12 364+10 367+16 385 _+14 354 + 19 363_+15 362+12

Values represent mean revertants per plate + standard error. TR, toxic response. * Maximum mutagenic potency. a Sodium nitrite (300/zg), pH 3.

TA100, a 2-fold increase (299 + 9) in the number of revertants was induced by 25 mg equivalent N-ATE. Metabolic activation led to a further increase in mutagenicity, which was maximum at 75 mg (360 + 4). N - A T E was non-mutagenic in strain TA102 in the presence and absence of $9.

Nitrosated aqueous: ethanolic bidi tobacco extract (N-AETE) N - A E T E was tested in the range 5 /xg to 200 mg equivalent. It was non-mutagenic in strain TA98 in the presence and absence of $9 (Table

2). In strain TA100, N - A E T E showed a linear dose response accompanied by a marginal increase (63%) in the number of revertants at 10 mg, which was abolished after addition of $9. In strain TA102, the nitrosated extract was highly mutagenic in the absence of $9 and induced an 8-fold increase (2454 + 31) over the spontaneous revertant number at 1 mg. Addition of $9 reduced the mutagenic potency but could not abolish it totally. Thus, a 3-fold increase over spontaneous revertants was induced by N - A E T E + $9 at 100 mg (969 _+ 18).

TABLE 2 M U T A G E N I C I T Y OF N I T R O S A T E D A Q U E O U S : E T H A N O L I C E X T R A C T OF BIDI TOBACCO Dose

TA98

(mg)

- $9

+ $9

TA100 - $9

+ $9

- $9

+ $9

0 a 0.5 1 5 10 50 100

29 + 2 25 + 3 26+1 28 + 3 27+2 TR TR

31 + 1 29+ 2 32+3 30 + 1 26+ 1 24 + 2 TR

130+ 1 135 + 3 169+4 185 + 4 207+4 b 162 + 4 TR

125 + 5 125 + 7 126+2 121 + 3 112+6 126 + 5 112 + 6

309+ 10 906+ 21 2454+31 * 1073 + 29 958+ 17 442 + 18 TR

310+ 345 + 412+ 450 + 442+ 655 + 969 +

Values represent mean revertants per plate + standard error. TR, toxic response. * Maximum mutagenic potency. a Sodium nitrite (300 txg), pH 3. b Weak response.

TA102

11 11 5 14 17 19 18 *

97 TABLE 3 MUTAGENICITY OF NITROSATED ETHANOLIC EXTRACT OF BIDI TOBACCO TA102

Dose (mg)

TA98 - $9

TA100 + $9

- $9

+ $9

- $9

+ $9

0a 5 10 25 50 100

29+-3 33+-3 34+-2 40+3 28+2 TR

304-_2 31 +-2 31 +2 30+3 33+_3 29 ++_2

125+- 3 155+- 10 237+- 18 372+- 15 * 214+- 15 TR

130-t-5 148 _+4 153_+7 111+-8 104+-6 TR

312+_ 11 824 + 10 1470+ 12 3839+-25 * 1871+-23 608+ 5

333_+ 10 392 + 14 417+- 18 467+-17 506+- 8 313+-11

Values represent mean revertants per plate + standard error. TR, toxic response. * Maximum mutagenic potency. a Sodium nitrite (300 ~g), pH 3.

Nitrosated ethanolic bidi tobacco extract (N-ETE) N - E T E was tested in the range 1-200 mg equivalent. As seen in Table 3, N - E T E was nonmutagenic in strain TA98, in the presence and absence of $9. In strain TA100 however, N - E T E exhibited maximum mutagenic potency at 25 mg, with a 3-fold increase (372 + 15) over the spontaneous reversion rate. While a d o s e - r e l a t e d response was noted in the absence of $9, addition of $9 r e n d e r e d the extract non-mutagenic. In strain TA102 again, N - E T E induced a dose-related response accompanied by a 12-fold increase in the n u m b e r of revertants at 25 mg (3839 + 25). A d d i t i o n of $9 r e n d e r e d the sample non-mutagenic.

Modified protocol for metabolic activation Table 4 reveals that in spite of the use of hamster liver $9 for metabolic activation, extrane-

TABLE 4 MUTAGENICITY TESTING OF AQUEOUS BIDI TOBACCO EXTRACT IN STRAIN TA100 a Dose (mg)

Revertants per plate (mean + SE)

0 5 25 50

127+ 5 172+ 10 191+ 9 * 179 + 13

a Modified protocol (Guttenplan, 1987a) using hamster liver $9. * 50% increase in number of revertants.

ous addition of N A D P H and weakly acidic p H of the incubation mix, the aqueous bidi tobacco extract was non-mutagenic in strain TA100. Discussion

The present study was initiated to d e t e r m i n e the mutagenicity of smokeless tobacco used for bidi manufacture, since workers employed in this industry receive large-scale cutaneous as well as nasopharyngeal exposure to coarse particles and fine respirable dust of tobacco. The results show that the aqueous, a q u e o u s : ethanolic, ethanolic and chloroform extracts of processed bidi tobacco in polar and non-polar solvents were non-mutagenic to TA98, TA100 and TA102 when tested in the presence and absence of metabolic activation. This is in contrast to the earlier reports of mutagenicity of an ethanolic extract of Indian chewing tobacco and aqueous extracts of snuff and chewing tobacco from the U.S.A. (Shah et al., 1985; Guttenplan, 1987a). While the ethanolic extract was mutagenic to TA98 in the presence of rat liver $9 (Shah et al., 1985), G u t t e n p l a n (1987a) r e p o r t e d that use of special assay conditions was required to d e m o n s t r a t e mutagenicity of aqueous tobacco extracts in strain TA100. Considering that the assay conditions in the p r e s e n t study were similar to those in the earlier reports, non-mutagenicity of aqueous and ethanolic extracts of bidi tobacco a p p e a r to be r e l a t e d to differences in the composition and processing of the finished products tested.

98

Processed tobacco is known to contain over 2500 chemicals including alkaloids, amines, amides, free amino acids, phenols, phenolic acids, carboxylic acids, tobacco-specific nitrosamines (TSNA), etc. (IARC, 1985; Schmeltz and Hoffmann, 1977). Upon nitrosation, some of these are converted into directly mutagenic N-nitroso compounds (NOC) such as nitrosamides and a few nitrosamines, as well as promutagenic nitrosamines such as TSNA, that require metabolic activation for mutagenic effect (Guttenplan, 1987b; Hecht and Hoffmann, 1988; Tannenbaum, 1981). The mutagenicity of smokeless tobacco has been largely attributed to TSNA (Hecht et al., 1983). However, in the present study, aqueous extract of bidi tobacco was non-mutagenic under conditions favoring mutagenicity due to nitrosamines. Moreover, nitrosated bidi tobacco extracts exhibited mutagenicity to various tester strains in the absence of $9, indicating that NOC other than TSNA are responsible for the mutagenic effect. In the cases where N-ATE and NAETE exhibited increased mutagenicity to TA100 and TA102 in the presence of $9 (Tables 1 and 2), it could be attributed to removal of toxic components, as clear-cut toxicity was evident at the doses tested in the absence of metabolic activation. The mutagenicity of nitrosated extracts observed in this study is in keeping with the reports of Whong et al. (1984, 1985) who showed that nitrosation rendered tobacco extracts mutagenic to TA98 and TA100. However, this is the first report on mutagenicity of smokeless tobacco to strain TA102, indicating that tobacco contains precursors that upon nitrosation cause mutagenicity via oxidative damage. Several workers have suggested that oxidative damage plays an important role in the process of tumor promotion (Cerutti, 1988; Ames, 1989). Since tobacco habitu6s are reported to exhibit increased endogenous nitrosation (Nair et al., 1986), it is felt that further studies along these lines on bidi industry workers would help to understand the possible implications of occupational exposure to bidi tobacco. In conclusion, the present study shows that nitrosated extracts of bidi tobacco exhibit mutagenicity due to frameshifts, base-pair substitu-

tions and oxidative damage. The findings imply that bidi industry workers are potentially at high risk for genotoxic hazards due to occupational exposure to bidi tobacco.

Acknowledgements The authors are grateful to Kiran Bidi Udyog Ltd. (Nipani, India) for providing bidi tobacco samples, Dr. U.K. Ganu for preparation of aqueous:ethanolic and chloroform extracts of tobacco, Mr. M. D'Mello and Mr. M.L. Jagtap for technical assistance, and Mr. D.C. Sequeira for typing the manuscript.

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