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Effect of tobacco extract and N′-nitrosonornicotine on the carcinogen metabolising enzymes under different dietary vitamin B status
153
Cancer Letters, 52 (1990) 153-159 Elsevier Scientific Publishers Ireland Ltd.
Effect of tobacco extract and Wnitrosonornicotine on the carcinogen metabolising enzymes under different dietary vitamin B status N. Ammigan”,
U.J. Nair”, A.J. Amonkarb
“Carcinogenesis Division and bBio-organic 400 012 (India)
Unit, Cancer
and S.V. Bhide” Research
Institute,
Tata Memorial Centte, Parer, Bombay
(Received 8 May 1989) (Revision received 16 May 1990) (Accepted 18 April 1990)
Summary
Introduction
Studies were carried out to eoaluate the changes in the phase I and !I enzymes of xenobiotic metabolism, on treatment with tobacco extract (TEI and a tobacco specific carcinogen, IV’-nittosonornicotine (NNN) in Sprague - Dawley rats maintained on vitamin B complex sufficient and deficient semi-synthetic diets. Both TE and NNN significantly increased the hepatic and pulmonary phase 1 enzymes in the vitamin B sufficient (SB’) and deficient (SB-) animals. However, the percent increase in enzyme activities was drastically higher in the SB- treated group as compared to those in the %+-treated group. On the other hand, TE and NNN significantly depressed the Leer and lung glutathione (GSH) level and glutathione S-transferase (GST) actiuiry in the SB- animals, while the opposite effect was obserued in the SB+treated animals. Furthermore, both the treatments depleted the hepatic pool of oitamin A, with a concurrent increase in that of vitamin C in SB’ and SB- groups.
Tobacco is probably the most important known carcinogen for human society today [ll]. In India, consumption of smokeless tobacco such as chewing tobacco, masheri (a pyrolysed tobacco product) and snuff is widespread [22] and is implicated in the high incidence of oropharyngeal cancer in this region [21]. Amongst the carcinogens present in tobacco and tobacco smoke are the tobacco specific N-nitrosamines (TSNA) N’nitrosonornicotine (NNN) and 4-(methylnitrosamine)-l-(3-pyridyl)-1-butanone (NNK) [2]. We have earlier reported high concentrations of TSNAs in raw and cured tobacco [ 11. NNN has been shown to cause oesophageal and nasal cavity tumours in rats [14] tracheal tumours in hamsters [13] and lung adenomas in mice [9]. TSNAs are known to be metabolised by the carcinogen metabolising enzymes to yield reactive intermediates which bind to various cellular macromolecules like DNA and proteins [4, lo]. These metabolising enzymes are known to be affected by nutritional factors. Amongst the various prevalent nutritional deficiencies in India, one of the most common is vitamin B
Keywords:
vitamin B; tobacco extract; N’nitrosonornicotine; metabolising enzymes. .
0304-3835/90/$03.50 Published and Printed
0 1990 Elsevier Scientiffc Publishers in Ireland
Ireland Ltd
154
complex deficiency [25,27]. The modulatory role of nutrient deficiencies and excess in the causation and prevention of cancer is well documented [17,32]. Hence, this study was undertaken to gain an insight in to the role of vitamin B complex on the metabolism of tobacco extract and NNN.
acid, 10 mg; folic acid, 1 mg; cyanocobalmin, 5 pg; biotin, 1 pg. This mixture was made up to 20 g with starch and used in the diet at 1%. SB- diet was similar to SB+ diet except that the vitamin mixture and choline chloride were replaced with starch.
Materials and methods
Preparation of tobacco extract TE was prepared by shaking 100 g of Pandharpuri tobacco (sun-dried) with 1 1 of dichloromethane at room temperature for 72 h. The extract was filtered and dried under vacua. The extract was then dissolved in dimethylsulphoxide at appropriate concentration .
Chemicals
All chemicals used were of the highest purity commercially available. Benzo[a] pyrene (BaP) , 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB) and l-chloro 2,4-dinitro benzene (CDNB) were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. 3Hydroxy benzo[a]pyrene (OH BaP) was a generous gift from Dr. J.N. Keith, IIT Research Institute, Chicago, IL, U.S.A. IV’Nitrosonornicotine (NNN) was synthesized and purified in our laboratory according to the method reported by Hu et al. [15]. Animals Inbred male weanling Sprague-Dawley rats (19-21 days) were obtained from the animal colony of the Cancer Research Institute, Bombay. Animals weighing 35-50 g were randomly divided into 2 groups. Group I was fed a semi-synthetic based diet (SB’) while Group II was fed the same diet but deficient in the B vitamins (SB-), 4-5 animals per cage were kept in a room with temperature maintained at 21 * l°C, humidity 50-55% and a 12-h light cycle. Composition ofsemi-synthetic based diet (g W) (SB”) Casein, 20; starch, 39.8; dextrose, 30; refined peanut oil, 5; salt mixture USP IV, 4; choline chloride, 0.2; vitamin mixture, 1; retinyl acetate, 20 IU. Composition of uitamin mixture Thiamine HCI, 41 mg; riboflavin, 4 mg; pyridoxine HCl, 1 mg; choline chloride, 750 mg; inositol, 200 mg; para-amino benzoic
Vitamin estimation Vitamins A and C were assayed as reported by Roels and Mahadevan [29] and Roe and Kurther [28], respectively. Riboflavin was assayed by a fluorometric method as reported by Loy et al. [19]. Folic acid was estimated by a microbiological method [12] using Lactobacillus casei ATCC 7469 as test organism. Experimental design During the experiment, body weights were determined in each group, at weekly intervals. At 12 weeks, the LD,, dose of TE and NNN were determined for each group. A total of 3/4 LD,, of TE and NNN were injected i-p. in 3 doses with 24-h intervals between each injection. Animals were fasted 24 h after the last injection. They were killed by cervical dislocation and the livers and lungs perfused with chilled potassium chloride solution (1.15%). The tissues were excised, rinsed in the above solution and homogenized in 4 volumes of ice-cold 0.25 M sucrose in 0.01 M phosphate buffer pH 7.4. The homogenate was centrifuged at 12,000 x g for 30 min. The resulting supernatant was re-centrifuged at 100,000 x g for 60 min. The microsomal pellet was suspended in 0.25 M sucrose in 0.01 M phosphate buffer pH 7.4. All tissue manipulations were carried out at 4OC.
155
Enzyme
assays
Microsomal cytochrome b and P-450 were determined by the method of Omura and Sato [25] using extinction coefficient of 171 cm-* mM-’ and 91 cm-* mM-‘, respectively. BaP hydroxylase activities were assayed as reported by Dehnen et al. [6]. Benzphetamine demethylase was assayed by the method of Lu et al. [20]. Reduced glutathione (GSH) was estimated as reported by Moron et al. [24] and glutathione S-transferase activity was assayed in the cytosol using CDNB and GSH as substrates [8]. Protein was estimated by Lowry’s method using bovine serum albumin as standard [18]. Statistical
analysis
Statistical analyses of data were carried out using Student’s t-test. Results
The total body weights in Sprague-Dawley rats at 12 weeks was not affected by vitamin B complex sufficient (SB’) or deficient (SB-) diet. However, significant decrease in liver ( - 38%) and lung ( - 31%) weights was observed in the SB- group. Riboflavin and folic acid levels ascertained as indicators of vitamin B status were drastically lowered in both liver and serum of the SB- group. Besides, the tolerance to TE/NNN was also decreased in the deficient group as reflected by the decreased LD,, dose in SB- group (Table 1). The basal hepatic and pulmonary cytochrome P-450 levels were significantly lower in the SB- group as compared to the SB+ group. The pulmonary values in untreated groups are lower than those in liver tissue of the corresponding group. Upon TE/NNN treatment, significant increase in hepatic cyt P-450 levels was observed in both SB’ and SB- groups. In lung tissue no change was observed in treated SB’ groups while SBgroup showed high pulmonary induction of Cyt P-450 upon treatment with TE ( + 225%) or NNN ( + 75%) (Table 2). Although the
basal levels of hepatic and pulmonary cytochrome b, were lower in the deficient group, NNN treatment did not affect cytochrome b, levels.
The basal levels of BaP hydroxylase and benzphetamine demethylase were significantly lower in the lung and liver of the SB- group. Upon treatment with TE/NNN a significant increase in both these enzymes was observed in SB+ and SB- groups. It was interesting to note that the percentage increase in enzyme was uniformly higher in the lungs than those in liver tissues of the SB- TE/NNN treated groups. Glutathione (GSH) levels were marginally lower in the SB’ group than those in the SB+ group. Significant increase was observed in hepatic as well as pulmonary GSH levels in treated SB’ groups while the levels of GSH were decreased in the SB- treated groups (Table 3). Basal GST activities in the SBgroup were lower in hepatic and pulmonary tissues as compared to those in the SB’ group. Upon treatment with TE/NNN, GST levels increased in the SB+ group while in the SB- group GST levels were significantly reduced (Table 3). Hepatic vitamin A and C levels were not affected by the vitamin B status of the animal. TE/NNN treatment resulted in significant decrease in vitamin A levels with a concurrent increase in vitamin C levels in both SB’ as well as SB- groups (Table 4). Discussion
Various workers have reported the protective effects of vitamins of the B-complex group. A daily dietary supplement of 200 c(9 of vitamin B, resulted iti a marked inhibition of liver tumours [5]. Chromosomal damage to human cells in vivo as a result of folate or vitamin B, deficiency has been reported to be extensive f231. To study the effect of vitamin B status on carcinogen metabolising enzymes, we found Sprague- Dawley rats ideal, as vitamin B deficiency was achieved by 12 weeks through the animals were put on SB-
(9)
8.3 + 0.8 5.1 + 0.3’
groups
SB’ SB-
1.3 f 0.6 0.67 f 0.3’
(9)
Lung wt.
0.48
SB-
235 f
SB-
0.38
SB-
+ 0.04
0.64 + 0.1
SB’
18
398 + 8
SB+
f 0.08
+ 0.06
(98%)
1.1 + 0.04’ (72%) 0.75 f 0.08’
(90%) 0.82 f 0.07’ (115%)
(73%)
1.21 + 0.12’
(74%)
(13%) 407 + 13’
14
450 f
(22%) 410 f
14
0.99 f 0.06’ + (31%) 1.1 + 0.05’ (130%)
486 -+ 16
(144%)
1.01 & 0.04’ + (34%) 1.17 + 0.06’
9
0.21 + 0.07
0.34 + 0.05
132 +
205 f 9
0.16 + 0.02
(24%) 0.57 + 0.08’ (171%)
0.42 f 0.07
(58%) 315 f 18’ (138%)
325 + 14’
(17%) 0.52 + 0.06’ (225%)
0.27 f 0.05
TE
12.5 f 1.7 4.7 f 0.4’
0.23 f 0.04
Results are mean f S.E. of 8 animals. *Values are significant compared to corresponding control groups at P < 0.001, n = 8. “Values are significant compared to SB’ control group (P < 0.005, n = 8). HCHO = formaldehyde. Values in parenthesis represent percent increase in enzyme activities from corresponding control groups.
Benzphetamine demethylase (nmol HCHO formed/min per mg protein)
BaP hydroxylase (pmol OHBaP formed/min per mg protein)
0.76
NNN
Control
TE
Control
n = 8).
48.5 f 2.4 0.5 + 0.1’
Pulmonary
SB+
7.3 + 0.4 0.5 + 0.3’
values in SB+ group (P < 0.001,
10.7 + 2.1 0.7 f 0.3’
Folic acid 1.19/g
Riboflavin ccg/g
Folic acid pg/ml
Riboflavin
Liver
pgC1mt
Serum
Hepatic
Cytochrome P-450 (nmol/mg protein)
on the phase I enzymes.
Dietary regimen
Effect of TE/NNN
Parameters
Table 2.
Results are mean f SE. of 8 animals. *Statistically significant as compared to corresponding
Liver wt.
Effect of dietary vitamin B on various parameters.
Dietary
Table 1.
245 190
NNN
(20%) 0.5 f 0.04’ (138%)
0.44 f 0.06
(54%) 301 + 12’ (128%)
317 -e 12’
0.30 f 0.04 (175%) 0.44 f 0.07’ (175%)
NNN
325 200
TE
LD,, (mg/kg)
k
SB-
SB+
(nmol CDNB conjugated per min per mg protein)
Glutathione (nmol/g tissue)
14
3.62
k 0.11”
3.9 -+ 0.04
429 -+ 19
585 f
4.39 * 0.2’ + (12.6%) 2.18 + 0.1’ - (39%)
control group.
4.36 -+ 0.1’ + (12%) 2.38 + 0.06’ - (37%)
623 + 15 + (6%) 320 + 7’ - (25%)
632 k 14’ + (8%) 310 f 11’ - (27%)
Results are mean + S.E. of 8 animals. *Values are significant compared to corresponding control group. P < 0.001, n = 8. ‘Values are significant compared to SB’ control group at (P < 0.005, n = 8). Values in parenthesis represent percent increase ( + ) decrease ( - ) from corresponding
SB-
SB+
Glutathione-S Transferase 2
6
0.99
+ 0.04
1.01 f 0.04
59+
132 f
Control
NNN
Control
system. Pulmonary
TE
detoxification
Hepatic
on the glutathione
Dietary regimen
Effect of TE/NNN
Parameters
Table 3.
1.31 -+ 0.07’ + (29%) 0.78 f 0.08 - (21%)
160 + 4’ + (21%) 30+ 3’ - (49%)
TE
1.28 k 0.08’ + (26%) 0.77 f 0.07 - (22%)
164 + 3’ + (24%) 45k 8’ - (24%)
NNN
158
Table 4.
Dietary regimen
Effect of TE/NNN on the hepatic pools of vitamin A and C. Vitamin A (pg/g)
Vitamin C (pg/g)
Control
TE
NNN
Control
TE
NNN
SB’
352 f 17 375 f 14
242 f 15 - (31%) 238 k 5’ - (36%)
277 * 10
SB-
230 ztz5’ - (34%) 215 f 3’ - (42%)
337 + 14’ + (21%) 343 + 7’ + (31%)
320 * 12’ + (15%) 390 -+ 10’ + (30%)
261+
5
Results are mean * S.E. of 8 animals. ‘Values are significant compared to corresponding control groups at P < 0.001, n = 8. Values in parenthesis represent percent increase ( + ), decrease ( - ) from corresponding control groups.
diet only after weaning, otherwise the animals remained apparently healthy. Our studies showed that the basal levels of cytochromes, P-450 and b, BaP hydroxylase and benzphetamine demethylase, were 30-40% lower in the SB- group as compared to the SB’ group in liver and lung (Table 2). While the GSH levels were marginally affected in liver and lung, GST levels were drastically reduced in the lung (- 55%) of the deficient group as compared to -26% in the SB’ group. The B-vitamins are of special interest in relation to metabolising enzyme functions because of their direct involvement as cofactors viz. niacin as NADPH, riboflavin as parts of the prosthetic group of reductase. Pyridoxine is reported to be involved in the pathway of heme synthesis and subsequently in synthesis of cytochrome P-450 [7]. Upon the treatment with TE/NNN, cytochrome P-450, BaP hydroxylase and benzphetamine demethylase levels were increased in lung and liver of both SB’ and SB- groups. The induced levels in the deficient group reached the basal levels in the SB’ group, the percentage increase being highest in the lungs of the deficient group. On the other hand GSH and GST which function as a possible major protective mechanism against genotoxic effects of many metabolically activated ultimate carcinogens, showed an interesting pattern of modulation [3]. While the GSH/GST system was induced in SB’ groups upon treatment with TE/NNN, a drastic depletion
in GST/GSH levels was observed in the deficient SB- group. Jernstrom et al. [16] have shown a reduction of intracellular GSH to about 40% of the normal levels with a concurrent 2 fold increase in DNA binding of BaP metabolites. The fine balance between activating and deactivating enzymes may be an important factor in determining the availability of electrophilic species for interaction with macromolecules. Our results clearly demonstrate that vitamin B complex deficiency has a differential effect on the inducibility of various metabolising enzymes, in the lung and liver. The effects are more pronounced in the lung, a target tissue for NNN [9]. In addition, we also found a decrease in vitamin A levels in both the groups upon treatment with TE/ NNN. Low levels of vitamin A have been found to be associated with increased risk of bronchogenic carcinoma [30] and oral cancer [31]. Thus, our studies indicate that vitamin B status could play an important role in the susceptibility to tobacco exposure, directly by its effect on carcinogen metabolising enzymes and indirectly by depletion in vitamin A levels. References Bhide, S.V., Nair, J., Maru, G.B., Nair, U.J., Rao, K.V.K., Chakraborty, M.K. and Brunnemann, K.D. (1987) Tobacco specific nitrosamines in green mature and processed tobacco leaves horn India. Be&age, Tabak. Forschung, Int., 14, 1, 29-32. Castonguay, A., Foiles, P.G., Trushin, N. and Hecht, S.S. (1985) A study of DNA methylation by tobacco spe-
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F.W. (1966) Cytogenetic and cytochemical studies on marrow cells in B,, and folate deficiency. Blood, 28, 581 -594. Moron, M.S., De Pierce, J.W. and Mannervik, B. (1979)
genicity metabolism and possible role in human cancer. J. Environ. Health Sci., CII l-54. Hecht, S.S. and Hoffmann, D. (1988) Tobacco specific nitrosamines, an important group of carcinogen in
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tobacco and tobacco smoke. Carcinogenesis, 9, 875884. Herbert, V. and Bertino, J.R. (1967) In: The Vitamins 111, Chemistry, Physiology, Pathology. Methods, 2nd edn., pp. 243-276. Editors: P. Gyorgy and W.N. Pearson. Academic Press, New York. Hilfrich, J., Hecht, S.S. and Hoffmann, D. (1977) A study of tobacco carcinogenesis XV. Effects of N’nitroso-
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Cancer Letters, 52 (1990) 153-159 Elsevier Scientific Publishers Ireland Ltd.
Effect of tobacco extract and Wnitrosonornicotine on the carcinogen metabolising enzymes under different dietary vitamin B status N. Ammigan”,
U.J. Nair”, A.J. Amonkarb
“Carcinogenesis Division and bBio-organic 400 012 (India)
Unit, Cancer
and S.V. Bhide” Research
Institute,
Tata Memorial Centte, Parer, Bombay
(Received 8 May 1989) (Revision received 16 May 1990) (Accepted 18 April 1990)
Summary
Introduction
Studies were carried out to eoaluate the changes in the phase I and !I enzymes of xenobiotic metabolism, on treatment with tobacco extract (TEI and a tobacco specific carcinogen, IV’-nittosonornicotine (NNN) in Sprague - Dawley rats maintained on vitamin B complex sufficient and deficient semi-synthetic diets. Both TE and NNN significantly increased the hepatic and pulmonary phase 1 enzymes in the vitamin B sufficient (SB’) and deficient (SB-) animals. However, the percent increase in enzyme activities was drastically higher in the SB- treated group as compared to those in the %+-treated group. On the other hand, TE and NNN significantly depressed the Leer and lung glutathione (GSH) level and glutathione S-transferase (GST) actiuiry in the SB- animals, while the opposite effect was obserued in the SB+treated animals. Furthermore, both the treatments depleted the hepatic pool of oitamin A, with a concurrent increase in that of vitamin C in SB’ and SB- groups.
Tobacco is probably the most important known carcinogen for human society today [ll]. In India, consumption of smokeless tobacco such as chewing tobacco, masheri (a pyrolysed tobacco product) and snuff is widespread [22] and is implicated in the high incidence of oropharyngeal cancer in this region [21]. Amongst the carcinogens present in tobacco and tobacco smoke are the tobacco specific N-nitrosamines (TSNA) N’nitrosonornicotine (NNN) and 4-(methylnitrosamine)-l-(3-pyridyl)-1-butanone (NNK) [2]. We have earlier reported high concentrations of TSNAs in raw and cured tobacco [ 11. NNN has been shown to cause oesophageal and nasal cavity tumours in rats [14] tracheal tumours in hamsters [13] and lung adenomas in mice [9]. TSNAs are known to be metabolised by the carcinogen metabolising enzymes to yield reactive intermediates which bind to various cellular macromolecules like DNA and proteins [4, lo]. These metabolising enzymes are known to be affected by nutritional factors. Amongst the various prevalent nutritional deficiencies in India, one of the most common is vitamin B
Keywords:
vitamin B; tobacco extract; N’nitrosonornicotine; metabolising enzymes. .
0304-3835/90/$03.50 Published and Printed
0 1990 Elsevier Scientiffc Publishers in Ireland
Ireland Ltd
154
complex deficiency [25,27]. The modulatory role of nutrient deficiencies and excess in the causation and prevention of cancer is well documented [17,32]. Hence, this study was undertaken to gain an insight in to the role of vitamin B complex on the metabolism of tobacco extract and NNN.
acid, 10 mg; folic acid, 1 mg; cyanocobalmin, 5 pg; biotin, 1 pg. This mixture was made up to 20 g with starch and used in the diet at 1%. SB- diet was similar to SB+ diet except that the vitamin mixture and choline chloride were replaced with starch.
Materials and methods
Preparation of tobacco extract TE was prepared by shaking 100 g of Pandharpuri tobacco (sun-dried) with 1 1 of dichloromethane at room temperature for 72 h. The extract was filtered and dried under vacua. The extract was then dissolved in dimethylsulphoxide at appropriate concentration .
Chemicals
All chemicals used were of the highest purity commercially available. Benzo[a] pyrene (BaP) , 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB) and l-chloro 2,4-dinitro benzene (CDNB) were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. 3Hydroxy benzo[a]pyrene (OH BaP) was a generous gift from Dr. J.N. Keith, IIT Research Institute, Chicago, IL, U.S.A. IV’Nitrosonornicotine (NNN) was synthesized and purified in our laboratory according to the method reported by Hu et al. [15]. Animals Inbred male weanling Sprague-Dawley rats (19-21 days) were obtained from the animal colony of the Cancer Research Institute, Bombay. Animals weighing 35-50 g were randomly divided into 2 groups. Group I was fed a semi-synthetic based diet (SB’) while Group II was fed the same diet but deficient in the B vitamins (SB-), 4-5 animals per cage were kept in a room with temperature maintained at 21 * l°C, humidity 50-55% and a 12-h light cycle. Composition ofsemi-synthetic based diet (g W) (SB”) Casein, 20; starch, 39.8; dextrose, 30; refined peanut oil, 5; salt mixture USP IV, 4; choline chloride, 0.2; vitamin mixture, 1; retinyl acetate, 20 IU. Composition of uitamin mixture Thiamine HCI, 41 mg; riboflavin, 4 mg; pyridoxine HCl, 1 mg; choline chloride, 750 mg; inositol, 200 mg; para-amino benzoic
Vitamin estimation Vitamins A and C were assayed as reported by Roels and Mahadevan [29] and Roe and Kurther [28], respectively. Riboflavin was assayed by a fluorometric method as reported by Loy et al. [19]. Folic acid was estimated by a microbiological method [12] using Lactobacillus casei ATCC 7469 as test organism. Experimental design During the experiment, body weights were determined in each group, at weekly intervals. At 12 weeks, the LD,, dose of TE and NNN were determined for each group. A total of 3/4 LD,, of TE and NNN were injected i-p. in 3 doses with 24-h intervals between each injection. Animals were fasted 24 h after the last injection. They were killed by cervical dislocation and the livers and lungs perfused with chilled potassium chloride solution (1.15%). The tissues were excised, rinsed in the above solution and homogenized in 4 volumes of ice-cold 0.25 M sucrose in 0.01 M phosphate buffer pH 7.4. The homogenate was centrifuged at 12,000 x g for 30 min. The resulting supernatant was re-centrifuged at 100,000 x g for 60 min. The microsomal pellet was suspended in 0.25 M sucrose in 0.01 M phosphate buffer pH 7.4. All tissue manipulations were carried out at 4OC.
155
Enzyme
assays
Microsomal cytochrome b and P-450 were determined by the method of Omura and Sato [25] using extinction coefficient of 171 cm-* mM-’ and 91 cm-* mM-‘, respectively. BaP hydroxylase activities were assayed as reported by Dehnen et al. [6]. Benzphetamine demethylase was assayed by the method of Lu et al. [20]. Reduced glutathione (GSH) was estimated as reported by Moron et al. [24] and glutathione S-transferase activity was assayed in the cytosol using CDNB and GSH as substrates [8]. Protein was estimated by Lowry’s method using bovine serum albumin as standard [18]. Statistical
analysis
Statistical analyses of data were carried out using Student’s t-test. Results
The total body weights in Sprague-Dawley rats at 12 weeks was not affected by vitamin B complex sufficient (SB’) or deficient (SB-) diet. However, significant decrease in liver ( - 38%) and lung ( - 31%) weights was observed in the SB- group. Riboflavin and folic acid levels ascertained as indicators of vitamin B status were drastically lowered in both liver and serum of the SB- group. Besides, the tolerance to TE/NNN was also decreased in the deficient group as reflected by the decreased LD,, dose in SB- group (Table 1). The basal hepatic and pulmonary cytochrome P-450 levels were significantly lower in the SB- group as compared to the SB+ group. The pulmonary values in untreated groups are lower than those in liver tissue of the corresponding group. Upon TE/NNN treatment, significant increase in hepatic cyt P-450 levels was observed in both SB’ and SB- groups. In lung tissue no change was observed in treated SB’ groups while SBgroup showed high pulmonary induction of Cyt P-450 upon treatment with TE ( + 225%) or NNN ( + 75%) (Table 2). Although the
basal levels of hepatic and pulmonary cytochrome b, were lower in the deficient group, NNN treatment did not affect cytochrome b, levels.
The basal levels of BaP hydroxylase and benzphetamine demethylase were significantly lower in the lung and liver of the SB- group. Upon treatment with TE/NNN a significant increase in both these enzymes was observed in SB+ and SB- groups. It was interesting to note that the percentage increase in enzyme was uniformly higher in the lungs than those in liver tissues of the SB- TE/NNN treated groups. Glutathione (GSH) levels were marginally lower in the SB’ group than those in the SB+ group. Significant increase was observed in hepatic as well as pulmonary GSH levels in treated SB’ groups while the levels of GSH were decreased in the SB- treated groups (Table 3). Basal GST activities in the SBgroup were lower in hepatic and pulmonary tissues as compared to those in the SB’ group. Upon treatment with TE/NNN, GST levels increased in the SB+ group while in the SB- group GST levels were significantly reduced (Table 3). Hepatic vitamin A and C levels were not affected by the vitamin B status of the animal. TE/NNN treatment resulted in significant decrease in vitamin A levels with a concurrent increase in vitamin C levels in both SB’ as well as SB- groups (Table 4). Discussion
Various workers have reported the protective effects of vitamins of the B-complex group. A daily dietary supplement of 200 c(9 of vitamin B, resulted iti a marked inhibition of liver tumours [5]. Chromosomal damage to human cells in vivo as a result of folate or vitamin B, deficiency has been reported to be extensive f231. To study the effect of vitamin B status on carcinogen metabolising enzymes, we found Sprague- Dawley rats ideal, as vitamin B deficiency was achieved by 12 weeks through the animals were put on SB-
(9)
8.3 + 0.8 5.1 + 0.3’
groups
SB’ SB-
1.3 f 0.6 0.67 f 0.3’
(9)
Lung wt.
0.48
SB-
235 f
SB-
0.38
SB-
+ 0.04
0.64 + 0.1
SB’
18
398 + 8
SB+
f 0.08
+ 0.06
(98%)
1.1 + 0.04’ (72%) 0.75 f 0.08’
(90%) 0.82 f 0.07’ (115%)
(73%)
1.21 + 0.12’
(74%)
(13%) 407 + 13’
14
450 f
(22%) 410 f
14
0.99 f 0.06’ + (31%) 1.1 + 0.05’ (130%)
486 -+ 16
(144%)
1.01 & 0.04’ + (34%) 1.17 + 0.06’
9
0.21 + 0.07
0.34 + 0.05
132 +
205 f 9
0.16 + 0.02
(24%) 0.57 + 0.08’ (171%)
0.42 f 0.07
(58%) 315 f 18’ (138%)
325 + 14’
(17%) 0.52 + 0.06’ (225%)
0.27 f 0.05
TE
12.5 f 1.7 4.7 f 0.4’
0.23 f 0.04
Results are mean f S.E. of 8 animals. *Values are significant compared to corresponding control groups at P < 0.001, n = 8. “Values are significant compared to SB’ control group (P < 0.005, n = 8). HCHO = formaldehyde. Values in parenthesis represent percent increase in enzyme activities from corresponding control groups.
Benzphetamine demethylase (nmol HCHO formed/min per mg protein)
BaP hydroxylase (pmol OHBaP formed/min per mg protein)
0.76
NNN
Control
TE
Control
n = 8).
48.5 f 2.4 0.5 + 0.1’
Pulmonary
SB+
7.3 + 0.4 0.5 + 0.3’
values in SB+ group (P < 0.001,
10.7 + 2.1 0.7 f 0.3’
Folic acid 1.19/g
Riboflavin ccg/g
Folic acid pg/ml
Riboflavin
Liver
pgC1mt
Serum
Hepatic
Cytochrome P-450 (nmol/mg protein)
on the phase I enzymes.
Dietary regimen
Effect of TE/NNN
Parameters
Table 2.
Results are mean f SE. of 8 animals. *Statistically significant as compared to corresponding
Liver wt.
Effect of dietary vitamin B on various parameters.
Dietary
Table 1.
245 190
NNN
(20%) 0.5 f 0.04’ (138%)
0.44 f 0.06
(54%) 301 + 12’ (128%)
317 -e 12’
0.30 f 0.04 (175%) 0.44 f 0.07’ (175%)
NNN
325 200
TE
LD,, (mg/kg)
k
SB-
SB+
(nmol CDNB conjugated per min per mg protein)
Glutathione (nmol/g tissue)
14
3.62
k 0.11”
3.9 -+ 0.04
429 -+ 19
585 f
4.39 * 0.2’ + (12.6%) 2.18 + 0.1’ - (39%)
control group.
4.36 -+ 0.1’ + (12%) 2.38 + 0.06’ - (37%)
623 + 15 + (6%) 320 + 7’ - (25%)
632 k 14’ + (8%) 310 f 11’ - (27%)
Results are mean + S.E. of 8 animals. *Values are significant compared to corresponding control group. P < 0.001, n = 8. ‘Values are significant compared to SB’ control group at (P < 0.005, n = 8). Values in parenthesis represent percent increase ( + ) decrease ( - ) from corresponding
SB-
SB+
Glutathione-S Transferase 2
6
0.99
+ 0.04
1.01 f 0.04
59+
132 f
Control
NNN
Control
system. Pulmonary
TE
detoxification
Hepatic
on the glutathione
Dietary regimen
Effect of TE/NNN
Parameters
Table 3.
1.31 -+ 0.07’ + (29%) 0.78 f 0.08 - (21%)
160 + 4’ + (21%) 30+ 3’ - (49%)
TE
1.28 k 0.08’ + (26%) 0.77 f 0.07 - (22%)
164 + 3’ + (24%) 45k 8’ - (24%)
NNN
158
Table 4.
Dietary regimen
Effect of TE/NNN on the hepatic pools of vitamin A and C. Vitamin A (pg/g)
Vitamin C (pg/g)
Control
TE
NNN
Control
TE
NNN
SB’
352 f 17 375 f 14
242 f 15 - (31%) 238 k 5’ - (36%)
277 * 10
SB-
230 ztz5’ - (34%) 215 f 3’ - (42%)
337 + 14’ + (21%) 343 + 7’ + (31%)
320 * 12’ + (15%) 390 -+ 10’ + (30%)
261+
5
Results are mean * S.E. of 8 animals. ‘Values are significant compared to corresponding control groups at P < 0.001, n = 8. Values in parenthesis represent percent increase ( + ), decrease ( - ) from corresponding control groups.
diet only after weaning, otherwise the animals remained apparently healthy. Our studies showed that the basal levels of cytochromes, P-450 and b, BaP hydroxylase and benzphetamine demethylase, were 30-40% lower in the SB- group as compared to the SB’ group in liver and lung (Table 2). While the GSH levels were marginally affected in liver and lung, GST levels were drastically reduced in the lung (- 55%) of the deficient group as compared to -26% in the SB’ group. The B-vitamins are of special interest in relation to metabolising enzyme functions because of their direct involvement as cofactors viz. niacin as NADPH, riboflavin as parts of the prosthetic group of reductase. Pyridoxine is reported to be involved in the pathway of heme synthesis and subsequently in synthesis of cytochrome P-450 [7]. Upon the treatment with TE/NNN, cytochrome P-450, BaP hydroxylase and benzphetamine demethylase levels were increased in lung and liver of both SB’ and SB- groups. The induced levels in the deficient group reached the basal levels in the SB’ group, the percentage increase being highest in the lungs of the deficient group. On the other hand GSH and GST which function as a possible major protective mechanism against genotoxic effects of many metabolically activated ultimate carcinogens, showed an interesting pattern of modulation [3]. While the GSH/GST system was induced in SB’ groups upon treatment with TE/NNN, a drastic depletion
in GST/GSH levels was observed in the deficient SB- group. Jernstrom et al. [16] have shown a reduction of intracellular GSH to about 40% of the normal levels with a concurrent 2 fold increase in DNA binding of BaP metabolites. The fine balance between activating and deactivating enzymes may be an important factor in determining the availability of electrophilic species for interaction with macromolecules. Our results clearly demonstrate that vitamin B complex deficiency has a differential effect on the inducibility of various metabolising enzymes, in the lung and liver. The effects are more pronounced in the lung, a target tissue for NNN [9]. In addition, we also found a decrease in vitamin A levels in both the groups upon treatment with TE/ NNN. Low levels of vitamin A have been found to be associated with increased risk of bronchogenic carcinoma [30] and oral cancer [31]. Thus, our studies indicate that vitamin B status could play an important role in the susceptibility to tobacco exposure, directly by its effect on carcinogen metabolising enzymes and indirectly by depletion in vitamin A levels. References Bhide, S.V., Nair, J., Maru, G.B., Nair, U.J., Rao, K.V.K., Chakraborty, M.K. and Brunnemann, K.D. (1987) Tobacco specific nitrosamines in green mature and processed tobacco leaves horn India. Be&age, Tabak. Forschung, Int., 14, 1, 29-32. Castonguay, A., Foiles, P.G., Trushin, N. and Hecht, S.S. (1985) A study of DNA methylation by tobacco spe-
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