Cancer Letters, 55 (1990)
83
83-88
Elsevier Scientific Publishers Ireland Ltd.
Effect of cigarette smoke inhalation on benzo[a]pyreneinduced lung carcinogenesis in vitamin A deficiency in the rat M.P. Gupta, K.L. Khanduja, Department
of Biophysics, Postgraduate
(Received 20 May 1990) (Revision received 18 September iAccepted 20 September 1990)
I.B. Koul and R.R. Sharma Institute of Medical Education
and Research,
Chandigarh
- 160012 (India)
1990)
Summary Vitamin A deficiency caused a significant increase (P < 0.05) in benzo[a]pyrene (BP)induced lung tumor incidence and tumor burden in male Wistar rats. Inhalation of cigarette smoke during initiation and post-initiation phases of carcinogenesis resulted in higher tumor burden as compared to the same observed in the animals exposed to cigarette smoke during the post-initiation phase only. Stimulation in tumor burden by cigarette smoke was increased further by vitamin A deficiency .
Keywords: Vitamin A deficiency; smoke, lung tumors; benzo[a]pyrene
Cigarette
terious effects of cigarette smoke is dependent on the type of tobacco and cigarette, duration of smoking, age at start of smoking, depth of inhalation etc. [3]. Beside these, the role of nutrition in carcinogenesis cannot be ruled out. In this regard, investigations on vitamin A have attracted particular attention because of its role in growth and differentiation of epithelial cells. Besides this, epidemiological and animal studies have found a positive correlation between low intake of vitamin A and an increased risk of lung cancer [4,5]. Earlier we have shown that vitamin A deficiency in rat stimulated BPinduced lung tumorigenesis by acting both at initiation and post-initiation phases of carcinogenesis [6]. In the present study we have investigated the influence of passive cigarette smoke inhalation on BP-induced lung carcinogenesis in situations of vitamin A deficiency.
Introduction Materials and Methods Epidemiological reports clearly implicate ciagarette smoking as one of the major causes of lung cancer [l-3]. The large inter-individual variation in the lung’s susceptibility.to deleCorrespondence to: K.L. Khanduga,
Department
Postgraduate
Education
Chandigarh
Institute
- 160012,
0304-3835/90/$03.50
of Medical India.
0
of Biophysics, and
Research,
Male Weanling Wistar rats (50-70 g) were taken from the institute’s Animal Colony. These were divided into 12 groups according to the experiment plan shown in Fig. 1. All the animals were housed in wire mesh cages and served with fresh tap water and casein-based vitamin A-free diet ad libitum as described ear-
1990 Elsevier Scientific Publishers Ireland Ltd
Published and Printed in Ireland
CONTROL. CONIROL
* CIG.
SMOKE
CONTROL
* Fe203.
CONTROL BP.
*
Fe20j
CON1 ROL * + BP Fef,f
SMOKE.
CONTROL
‘SMOKE’
BP.Fe2%
* SMOKE.
WT.
A
DEF
VII.
A
DEF
*
SMOKE, “17
A
DEF
.
Fe203 VIT
A
BP
FezOx
WT. A
DEF
DEF.
Fe.f+’ SMXE *BP~Fc24’
6 709
-
. BP,
SMOKE *VII.
A DEF: SMOKE
24 WEEKS
Fig. 1. (Cl) (El) (m) (aa)
Protocol for lung tumor induction studies. Vitamin A-free diet + 100 IV vitamin A twice/week; vitamin A-free diet + 700 IU vitamin A twice/week; cigarette smoke inhalation (5 cigarettes/h per day; vitamin A-free diet; (&A)intratracheal instillations.
lier [7]. Animals in control groups (I-VI) orally received 700 IU retinyl acetate in 0.2 ml olive oil twice per week, and animals in vitamin A-deficient groups (VII-XII) received 0.2 ml olive oil only until the 6th week of the study. Hepatic vitamin A status of the animals was checked after 6 weeks by the method of Dugan et al. [8]. After the 6th week, animals in the control groups continued to receive 700 IU retinyl acetate, whereas a dose of 100 II-Jof the vitamin, twice per week, was supplemented to animals in vitamin A deficient groups (VIIXII). Body growth and food consumption was recorded weekly. Hepatic vitamin A status of animals was again checked after 24 weeks when all the animals were sacrificed for tumor study. BP, purchased from Sigma Chemical Co., U.S.A. was adsorbed onto ferric oxide by grinding the mixture (1: 1) for 5-6 h with a glass mortar and pestle. The mixture was sus-
pended in sterilized isotonic saline (20 mg BP: Fe,0,/0.2 ml) and the suspension was steadily stirred during use. Animals were administered with 3 weekly doses of 20 mg of BP-Fe 0, mixture (Groups IV-VI and X-XII) or lb mg Fe,O, (Groups III and IX) during the 6th--8th week of the investigation in 0.2 ml saline. Animals (Groups VI and XII) were subjected to cigarette smoke from the 2nd to the 24th week of the study. For studying the effect of cigarette smoke inhalation on the post-initiation phase of BP-induced carcinogenesis, animals of groups V and XI were subjected to smoke inhalation during the lOth-24th week of the study. Rats of Groups II and VIII, and I and VII were exposed to cigarette smoke and air, respectively, from the lOth-20th week of the study. The procedure for cigarette smoke inhalation in rats has been described by us earlier [9]. In short, 12 animals at a time were exposed to the smoke from five commercial filter cigarettes (1.25 mg nicotine and 35 mg tar/cigarette) for 1 h/day in an inhalation chamber of total volume of 8.2 litres, with separate inlets for smoke and air. The animals were exposed to air for 5 min after exposure to the smoke from each cigarette. The air flow through the chamber was controlled to 14.0 l/min by a valve situated between the outlet of the chamber and the suction pump. Only a small portion of the air passed through the cigarette which completely burnt in 8 min. All the animals were killed under light ether anesthesia after 24 weeks. The lungs were observed for the presence of visible tumor nodules. The tissues were serially cut to find the deep situated nodules. The mean diameter of the tumor was the average of vernier caliper measurements in two directions at right angles to each other. A portion of tumor tissue was fixed in formalin saline and processed for optical microscopic examination by hematoxylin/ eosin staining. Statistical significance of the difference in tumor incidence among various groups was checked by the x*-test. Significance of changes in vitamin A levels was checked by the unpaired Student’s t-test.
85
Results Body growth remained unaffected in all the groups of animals throughout the study (Table I). In 6 weeks hepatic vitamin A (retinyl easters), mean +- S.D., decreased from 96 f 12.6 pg/g to an undetectable level due to vitamin A deficiency. After 24 weeks, hepatic vitamin A level in the deficient groups (5.6 & 0.7 pg/g) was significantly less (P < 0.001) than the amount detected in the control group (98 f 9.4pg/g). Tumors appeared as pale yellow nodules of varying sizes, localized in various lobes of lungs. They were squamous cell carcinomas with massive keratinization and nests of neoplastic cells as described by us earlier [5]. We did not get any other type of tumor than squamous cell carcinoma. Table II shows the effects of cigarette smoke inhalation on initiation and post-initiation phases of BP-induced lung carcinogenesis in the rat in vitamin A deficiency. No lung tumor was induced by vitamin A deficiency, cigarette smoke inhalation or instillation of Fe,O, either separately or in combination with the other two factors. Vitamin A deficiency caused a significant increase (P < 0.01) in the incidence of BP-induced tumors in the sham-exposed group, and in animals exposed to cigarette smoke during the post-initiation phase (Group XI) only or during initiation and post-initiation phases (Group XII). The tumor burden of 5mm tumors induced by the carcinogen was increased due to vitamin A deficiency. Inhalation of cigarette smoke during the post-initiation phase only in control group (Group V) caused an increase of 9% and 50% in tumor burden of > 5 mm and < 5 mm tumors, respectively. Following inhalation of cigarette smoke during initiation and post-initiation phases in the control animals (Group VI), tumor burden of > 5 mm tumors and < 5 mm tumors was increased by 75% and 175% respectively. In vitamin A deficiency (Group XI), inhalation of cigarette smoke during post-initiation phase caused a decrease in burden of > 5 mm tumors
from 1.83 to 1.67, but the burden of < 5 mm tumors was increased 3-fold. Inhalation of cigarette smoke during initiation and post-initiation phases in vitamin A deficient animals (Group XII) increased the combined tumor burden of > 5 mm and < 5 mm tumors to a relatively greater extent in comparison with the corresponding change observed in the vitamin A deficient group exposed to cigarette smoke during the post-initiation phase (Group XI) only. For example, total tumor burden due to cigarette smoke inhalation in initiation and post-initiation phases of BP-induced carcinogenesis in vitamin A deficiency was increased from 2.00 to 3.36 (Group X vs. XII); whereas cigarette smoke inhalation during the post-initiation phase increased the combined tumor burden from 2.00 to 2.17 (Group X vs. XI). Inhalation of cigarette smoke in vitamin A deficiency during the post-initiation phase (Group XI) of BP-induced tumorigenesis resulted in 34% increase in total tumor burden. Whereas the increase caused by vitamin A deficiency (Group X) or inhalation of cigarette smoke during post-initiation phase (Group V) only was 23.5% and 19.1%, respectively. Discussion In the present as well as in the earlier study [6] we have used the development of metaplastic lung nodules, induced by BP in rats as an indicator to the effects of vitamin A deficiency and/or cigarette smoke inhalation. A common feature of these studies is that the susceptibility to lung carcinogenesis was increased in spite of absence of any visible signs of vitamin A deficiency, which were avoided due to presence of considerable amount of plasma vitamin A store. An interesting aspect of this investigation is that BPinduced lung tumor burden was found to be increased to a much lesser extent in control and vitamin A-deficient animals exposed to cigarette smoke during post-initiation phase as compared to increase in the animals exposed to cigarette smoke during initiation and postinitiation phases. Thus, it appears that cigarette
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r(
5
=
X
35
35
35
34
3.5
34
28
24”
24”
28 (100)’
24 (loo)*
24 (100)’
l
0.50
0.50
0.17
These animals were included for calculations
2.86
1.67
1.83
0 0
0 (0)
18
18
25
0
0
0 (0)
10
1.1
0.6 2.14
1.33
0.4
10
10
23
12
1.22
0
0
0
47
26
8
0 0
< 5mm
0 0
> 5mm
Tumor burden
rats.
0
22 (79)
20 (77)
22
0
0
0 (0) 18 (69)
0 0
< 5mm
0 0
> 5mm
No. of tumors detected
control and vitamin A-deficient
0 (0) 0 (0)
No. of tumor bearing animals
BP-administered
0 (0)
10
20’
28
26
26”
19
10 10
No. of animals sacrificed for tumor i.t. instillation
in intratracheally
10
35
35
33
19
35
35
35
25
10 10
No. of animals survived after last
on lung tumor induction
Ten animals were killed to estimate hepatic vitamin A status after 6th week to confirm vitamin A deficicney. “Two animals in each of these groups died 15th or 16th week after i.t. instillation to possess lung tumors. of lung tumor burden. Values in parenthesis are the % tumor incidence. “P< 0.05: Groups V, VI, X and XII compared with Group IV.
XII
XI
X
IX
VIII
VII
VI
V
IV
III
20’ 10
I II
Control Control + smoke Control + Fe,O, Control + BR-Fe,O, Control + BP-Fe,O,-smoke Control + smoke + BP-Fe,O,-smoke Vitamin A def. Vitamin A def . + smoke Vitamin A def + Fe,O, Vitamin A def. + BP-Fe,O, Vitamin A def + BP-Fe,O,-smoke Vitamin A def. + smoke + BPFe,O,-smoke
Initial No. of animals
Effect of cigarette smoke inhalation
Group
Table II.
88
smoke inhalation caused greater stimulation in the carcinogenic process mainly by acting at the initiation phase of BP-induced carcinogenesis. While cigarette smoke condensate itself has been suggested to be a relatively poor tumor initiating agent in mouse skin [lo], results in the present study indicate that in combination with other environmental agents, like BP it can also act as a potential stimulator of the initiation process possibly through parameters involved in the regulation of chemical carcinogenesis. Certain alterations in the activity of carcinogen activating and detoxifying enzymes have been reported to occur in vitamin A deficiency [7,11]. The impairment in balance of activities of these enzymes due to decreased levels of carcinogen detoxifying enzymes appear to enhance covalent binding of the carcinogens to DNA [ 121, which is a critical step in expliciting the carcinogenic, mutagenic and toxic effects of the chemicals. Therefore, the observed enhancement in lung carcinogenesis in vitamin A deficiency could be attributable at least in part to impairment of carcinogen detoxifying enzymes activities in the lung [ 131. We have earlier observed that inhalation cigarette smoke enhances in vitro and in vivo covalent binding of [3H]BP to DNA. The extent of increase in binding was more in vitamin A deficient animals. Similarly, it found that inducibility of aryl hydrocarbon hydroxylase activity in lungs by cigarette smoke was more in vitamin A deficient rats. Therefore, it is likely that the stronger effect of cigarette smoke inhalation on the initiation phase of chemical carcinogenesis in vitamin A deficiency has the linkage with these changes in carcinogen metabolizing enzymes pattern. Acknowledgement A financial grant from the Indian Council of Medical Research, New Delhi is gratefully acknowledged.
References 1 2 3
4
Reif, A.E. (1958) International Cancer Congress Science. N.Y., 128, 1512-1522. Doll, R. (1978) An epidemiological perspective of the biology of cancer. Cancer Res., 38,3573-3583. IARC risk of 1986, Bjelke,
monographs on the evaluation of the carcinogenic chemicals to humans: Tobacco smoking. Vol. 38, Lyon, France. E. (1975) Dietary vitamin A and human lung can-
7
cer. Int. J. Cancer, 15,561-565. Mettlin, C.. Grahm, S. and Swanson, M. (1979) Vitamin A and lung cancer. J. Natl, Cancer Inst., 62, 1435-1438. Dogra, S.C., Khanduja, K.L. and Gupta, M.P. (1985). The effect of vitamin A deficiency on initiation and postinitiatfon phases of benzo(a)pyrene induced lung tumorigenesis in rat. Br. J. Cancer, 52, 931-935. Dogra, S.C., Khanduja, K.L., Gupta, M.P. and Sharma,
8
R.R. (1983). Effect of vitamin A deficiency on the pulmonary and hepatic drug metabolizing enzymes in rat. Enzymes, 30,99-104. Dugan, R.E., Frigerio, N.A. and Gilbert, J.M. (1964).
5 6
9
10
11
12
13
Calorimetric determination of vitamin A and its derivatives with trifluoroacetic acid. Anal. Chem., 36, 114-117. Gupta, M.P., Khanduja, K.L. and Sharma, R.R. (1988) Effect of cigarette smoke inhalation on antioxidant enzymes and lipid peroxidation in rat. Toxicol. Lett., 41, 107-114. Wynder, E.L. and Hoffman, D. (1964) Experimental tobacco carcinogenesis. In: Adv. Cancer Res., 8, 249453. Miranda, C.L., Mukhtar, H., Bend, J.R. and Chhabra, R.S. (1979) Effects of vitamin A deficiency on hepatic and extrahepatic mixed function oxidase and epoxide metabolizing enzymes in guinea pig and rabbit. Biochem. Pharmacol., 28, 2713-2716. Gupta, M.P., Khanduja, K.L. and Sharma, R.R. (1987) Effect of cigarette smoke inhalation on (3H) benzo(a)pyrene binding to lung DNA of vitamin A deficient rats. Med. Sci. Res., 15, 1323-1324. Gupta, M.P., Khanduja, K.L. and Sharma, R.R. (1986) Effect of cigarette smoke inhalation on certain pulmonary and hepatic drug metabolizing enzymes in rat in vitamin A deficiency. Indian J. Med. Res., 84, 301-309.
83
83-88
Elsevier Scientific Publishers Ireland Ltd.
Effect of cigarette smoke inhalation on benzo[a]pyreneinduced lung carcinogenesis in vitamin A deficiency in the rat M.P. Gupta, K.L. Khanduja, Department
of Biophysics, Postgraduate
(Received 20 May 1990) (Revision received 18 September iAccepted 20 September 1990)
I.B. Koul and R.R. Sharma Institute of Medical Education
and Research,
Chandigarh
- 160012 (India)
1990)
Summary Vitamin A deficiency caused a significant increase (P < 0.05) in benzo[a]pyrene (BP)induced lung tumor incidence and tumor burden in male Wistar rats. Inhalation of cigarette smoke during initiation and post-initiation phases of carcinogenesis resulted in higher tumor burden as compared to the same observed in the animals exposed to cigarette smoke during the post-initiation phase only. Stimulation in tumor burden by cigarette smoke was increased further by vitamin A deficiency .
Keywords: Vitamin A deficiency; smoke, lung tumors; benzo[a]pyrene
Cigarette
terious effects of cigarette smoke is dependent on the type of tobacco and cigarette, duration of smoking, age at start of smoking, depth of inhalation etc. [3]. Beside these, the role of nutrition in carcinogenesis cannot be ruled out. In this regard, investigations on vitamin A have attracted particular attention because of its role in growth and differentiation of epithelial cells. Besides this, epidemiological and animal studies have found a positive correlation between low intake of vitamin A and an increased risk of lung cancer [4,5]. Earlier we have shown that vitamin A deficiency in rat stimulated BPinduced lung tumorigenesis by acting both at initiation and post-initiation phases of carcinogenesis [6]. In the present study we have investigated the influence of passive cigarette smoke inhalation on BP-induced lung carcinogenesis in situations of vitamin A deficiency.
Introduction Materials and Methods Epidemiological reports clearly implicate ciagarette smoking as one of the major causes of lung cancer [l-3]. The large inter-individual variation in the lung’s susceptibility.to deleCorrespondence to: K.L. Khanduga,
Department
Postgraduate
Education
Chandigarh
Institute
- 160012,
0304-3835/90/$03.50
of Medical India.
0
of Biophysics, and
Research,
Male Weanling Wistar rats (50-70 g) were taken from the institute’s Animal Colony. These were divided into 12 groups according to the experiment plan shown in Fig. 1. All the animals were housed in wire mesh cages and served with fresh tap water and casein-based vitamin A-free diet ad libitum as described ear-
1990 Elsevier Scientific Publishers Ireland Ltd
Published and Printed in Ireland
CONTROL. CONIROL
* CIG.
SMOKE
CONTROL
* Fe203.
CONTROL BP.
*
Fe20j
CON1 ROL * + BP Fef,f
SMOKE.
CONTROL
‘SMOKE’
BP.Fe2%
* SMOKE.
WT.
A
DEF
VII.
A
DEF
*
SMOKE, “17
A
DEF
.
Fe203 VIT
A
BP
FezOx
WT. A
DEF
DEF.
Fe.f+’ SMXE *BP~Fc24’
6 709
-
. BP,
SMOKE *VII.
A DEF: SMOKE
24 WEEKS
Fig. 1. (Cl) (El) (m) (aa)
Protocol for lung tumor induction studies. Vitamin A-free diet + 100 IV vitamin A twice/week; vitamin A-free diet + 700 IU vitamin A twice/week; cigarette smoke inhalation (5 cigarettes/h per day; vitamin A-free diet; (&A)intratracheal instillations.
lier [7]. Animals in control groups (I-VI) orally received 700 IU retinyl acetate in 0.2 ml olive oil twice per week, and animals in vitamin A-deficient groups (VII-XII) received 0.2 ml olive oil only until the 6th week of the study. Hepatic vitamin A status of the animals was checked after 6 weeks by the method of Dugan et al. [8]. After the 6th week, animals in the control groups continued to receive 700 IU retinyl acetate, whereas a dose of 100 II-Jof the vitamin, twice per week, was supplemented to animals in vitamin A deficient groups (VIIXII). Body growth and food consumption was recorded weekly. Hepatic vitamin A status of animals was again checked after 24 weeks when all the animals were sacrificed for tumor study. BP, purchased from Sigma Chemical Co., U.S.A. was adsorbed onto ferric oxide by grinding the mixture (1: 1) for 5-6 h with a glass mortar and pestle. The mixture was sus-
pended in sterilized isotonic saline (20 mg BP: Fe,0,/0.2 ml) and the suspension was steadily stirred during use. Animals were administered with 3 weekly doses of 20 mg of BP-Fe 0, mixture (Groups IV-VI and X-XII) or lb mg Fe,O, (Groups III and IX) during the 6th--8th week of the investigation in 0.2 ml saline. Animals (Groups VI and XII) were subjected to cigarette smoke from the 2nd to the 24th week of the study. For studying the effect of cigarette smoke inhalation on the post-initiation phase of BP-induced carcinogenesis, animals of groups V and XI were subjected to smoke inhalation during the lOth-24th week of the study. Rats of Groups II and VIII, and I and VII were exposed to cigarette smoke and air, respectively, from the lOth-20th week of the study. The procedure for cigarette smoke inhalation in rats has been described by us earlier [9]. In short, 12 animals at a time were exposed to the smoke from five commercial filter cigarettes (1.25 mg nicotine and 35 mg tar/cigarette) for 1 h/day in an inhalation chamber of total volume of 8.2 litres, with separate inlets for smoke and air. The animals were exposed to air for 5 min after exposure to the smoke from each cigarette. The air flow through the chamber was controlled to 14.0 l/min by a valve situated between the outlet of the chamber and the suction pump. Only a small portion of the air passed through the cigarette which completely burnt in 8 min. All the animals were killed under light ether anesthesia after 24 weeks. The lungs were observed for the presence of visible tumor nodules. The tissues were serially cut to find the deep situated nodules. The mean diameter of the tumor was the average of vernier caliper measurements in two directions at right angles to each other. A portion of tumor tissue was fixed in formalin saline and processed for optical microscopic examination by hematoxylin/ eosin staining. Statistical significance of the difference in tumor incidence among various groups was checked by the x*-test. Significance of changes in vitamin A levels was checked by the unpaired Student’s t-test.
85
Results Body growth remained unaffected in all the groups of animals throughout the study (Table I). In 6 weeks hepatic vitamin A (retinyl easters), mean +- S.D., decreased from 96 f 12.6 pg/g to an undetectable level due to vitamin A deficiency. After 24 weeks, hepatic vitamin A level in the deficient groups (5.6 & 0.7 pg/g) was significantly less (P < 0.001) than the amount detected in the control group (98 f 9.4pg/g). Tumors appeared as pale yellow nodules of varying sizes, localized in various lobes of lungs. They were squamous cell carcinomas with massive keratinization and nests of neoplastic cells as described by us earlier [5]. We did not get any other type of tumor than squamous cell carcinoma. Table II shows the effects of cigarette smoke inhalation on initiation and post-initiation phases of BP-induced lung carcinogenesis in the rat in vitamin A deficiency. No lung tumor was induced by vitamin A deficiency, cigarette smoke inhalation or instillation of Fe,O, either separately or in combination with the other two factors. Vitamin A deficiency caused a significant increase (P < 0.01) in the incidence of BP-induced tumors in the sham-exposed group, and in animals exposed to cigarette smoke during the post-initiation phase (Group XI) only or during initiation and post-initiation phases (Group XII). The tumor burden of 5mm tumors induced by the carcinogen was increased due to vitamin A deficiency. Inhalation of cigarette smoke during the post-initiation phase only in control group (Group V) caused an increase of 9% and 50% in tumor burden of > 5 mm and < 5 mm tumors, respectively. Following inhalation of cigarette smoke during initiation and post-initiation phases in the control animals (Group VI), tumor burden of > 5 mm tumors and < 5 mm tumors was increased by 75% and 175% respectively. In vitamin A deficiency (Group XI), inhalation of cigarette smoke during post-initiation phase caused a decrease in burden of > 5 mm tumors
from 1.83 to 1.67, but the burden of < 5 mm tumors was increased 3-fold. Inhalation of cigarette smoke during initiation and post-initiation phases in vitamin A deficient animals (Group XII) increased the combined tumor burden of > 5 mm and < 5 mm tumors to a relatively greater extent in comparison with the corresponding change observed in the vitamin A deficient group exposed to cigarette smoke during the post-initiation phase (Group XI) only. For example, total tumor burden due to cigarette smoke inhalation in initiation and post-initiation phases of BP-induced carcinogenesis in vitamin A deficiency was increased from 2.00 to 3.36 (Group X vs. XII); whereas cigarette smoke inhalation during the post-initiation phase increased the combined tumor burden from 2.00 to 2.17 (Group X vs. XI). Inhalation of cigarette smoke in vitamin A deficiency during the post-initiation phase (Group XI) of BP-induced tumorigenesis resulted in 34% increase in total tumor burden. Whereas the increase caused by vitamin A deficiency (Group X) or inhalation of cigarette smoke during post-initiation phase (Group V) only was 23.5% and 19.1%, respectively. Discussion In the present as well as in the earlier study [6] we have used the development of metaplastic lung nodules, induced by BP in rats as an indicator to the effects of vitamin A deficiency and/or cigarette smoke inhalation. A common feature of these studies is that the susceptibility to lung carcinogenesis was increased in spite of absence of any visible signs of vitamin A deficiency, which were avoided due to presence of considerable amount of plasma vitamin A store. An interesting aspect of this investigation is that BPinduced lung tumor burden was found to be increased to a much lesser extent in control and vitamin A-deficient animals exposed to cigarette smoke during post-initiation phase as compared to increase in the animals exposed to cigarette smoke during initiation and postinitiation phases. Thus, it appears that cigarette
tl
cu
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5
tl
tl
+I
+I
-
0-Y
m
co
cj
+I
+I
= >
$
z
i
+I
+I
tl
X
R
r(
5
=
X
35
35
35
34
3.5
34
28
24”
24”
28 (100)’
24 (loo)*
24 (100)’
l
0.50
0.50
0.17
These animals were included for calculations
2.86
1.67
1.83
0 0
0 (0)
18
18
25
0
0
0 (0)
10
1.1
0.6 2.14
1.33
0.4
10
10
23
12
1.22
0
0
0
47
26
8
0 0
< 5mm
0 0
> 5mm
Tumor burden
rats.
0
22 (79)
20 (77)
22
0
0
0 (0) 18 (69)
0 0
< 5mm
0 0
> 5mm
No. of tumors detected
control and vitamin A-deficient
0 (0) 0 (0)
No. of tumor bearing animals
BP-administered
0 (0)
10
20’
28
26
26”
19
10 10
No. of animals sacrificed for tumor i.t. instillation
in intratracheally
10
35
35
33
19
35
35
35
25
10 10
No. of animals survived after last
on lung tumor induction
Ten animals were killed to estimate hepatic vitamin A status after 6th week to confirm vitamin A deficicney. “Two animals in each of these groups died 15th or 16th week after i.t. instillation to possess lung tumors. of lung tumor burden. Values in parenthesis are the % tumor incidence. “P< 0.05: Groups V, VI, X and XII compared with Group IV.
XII
XI
X
IX
VIII
VII
VI
V
IV
III
20’ 10
I II
Control Control + smoke Control + Fe,O, Control + BR-Fe,O, Control + BP-Fe,O,-smoke Control + smoke + BP-Fe,O,-smoke Vitamin A def. Vitamin A def . + smoke Vitamin A def + Fe,O, Vitamin A def. + BP-Fe,O, Vitamin A def + BP-Fe,O,-smoke Vitamin A def. + smoke + BPFe,O,-smoke
Initial No. of animals
Effect of cigarette smoke inhalation
Group
Table II.
88
smoke inhalation caused greater stimulation in the carcinogenic process mainly by acting at the initiation phase of BP-induced carcinogenesis. While cigarette smoke condensate itself has been suggested to be a relatively poor tumor initiating agent in mouse skin [lo], results in the present study indicate that in combination with other environmental agents, like BP it can also act as a potential stimulator of the initiation process possibly through parameters involved in the regulation of chemical carcinogenesis. Certain alterations in the activity of carcinogen activating and detoxifying enzymes have been reported to occur in vitamin A deficiency [7,11]. The impairment in balance of activities of these enzymes due to decreased levels of carcinogen detoxifying enzymes appear to enhance covalent binding of the carcinogens to DNA [ 121, which is a critical step in expliciting the carcinogenic, mutagenic and toxic effects of the chemicals. Therefore, the observed enhancement in lung carcinogenesis in vitamin A deficiency could be attributable at least in part to impairment of carcinogen detoxifying enzymes activities in the lung [ 131. We have earlier observed that inhalation cigarette smoke enhances in vitro and in vivo covalent binding of [3H]BP to DNA. The extent of increase in binding was more in vitamin A deficient animals. Similarly, it found that inducibility of aryl hydrocarbon hydroxylase activity in lungs by cigarette smoke was more in vitamin A deficient rats. Therefore, it is likely that the stronger effect of cigarette smoke inhalation on the initiation phase of chemical carcinogenesis in vitamin A deficiency has the linkage with these changes in carcinogen metabolizing enzymes pattern. Acknowledgement A financial grant from the Indian Council of Medical Research, New Delhi is gratefully acknowledged.
References 1 2 3
4
Reif, A.E. (1958) International Cancer Congress Science. N.Y., 128, 1512-1522. Doll, R. (1978) An epidemiological perspective of the biology of cancer. Cancer Res., 38,3573-3583. IARC risk of 1986, Bjelke,
monographs on the evaluation of the carcinogenic chemicals to humans: Tobacco smoking. Vol. 38, Lyon, France. E. (1975) Dietary vitamin A and human lung can-
7
cer. Int. J. Cancer, 15,561-565. Mettlin, C.. Grahm, S. and Swanson, M. (1979) Vitamin A and lung cancer. J. Natl, Cancer Inst., 62, 1435-1438. Dogra, S.C., Khanduja, K.L. and Gupta, M.P. (1985). The effect of vitamin A deficiency on initiation and postinitiatfon phases of benzo(a)pyrene induced lung tumorigenesis in rat. Br. J. Cancer, 52, 931-935. Dogra, S.C., Khanduja, K.L., Gupta, M.P. and Sharma,
8
R.R. (1983). Effect of vitamin A deficiency on the pulmonary and hepatic drug metabolizing enzymes in rat. Enzymes, 30,99-104. Dugan, R.E., Frigerio, N.A. and Gilbert, J.M. (1964).
5 6
9
10
11
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13
Calorimetric determination of vitamin A and its derivatives with trifluoroacetic acid. Anal. Chem., 36, 114-117. Gupta, M.P., Khanduja, K.L. and Sharma, R.R. (1988) Effect of cigarette smoke inhalation on antioxidant enzymes and lipid peroxidation in rat. Toxicol. Lett., 41, 107-114. Wynder, E.L. and Hoffman, D. (1964) Experimental tobacco carcinogenesis. In: Adv. Cancer Res., 8, 249453. Miranda, C.L., Mukhtar, H., Bend, J.R. and Chhabra, R.S. (1979) Effects of vitamin A deficiency on hepatic and extrahepatic mixed function oxidase and epoxide metabolizing enzymes in guinea pig and rabbit. Biochem. Pharmacol., 28, 2713-2716. Gupta, M.P., Khanduja, K.L. and Sharma, R.R. (1987) Effect of cigarette smoke inhalation on (3H) benzo(a)pyrene binding to lung DNA of vitamin A deficient rats. Med. Sci. Res., 15, 1323-1324. Gupta, M.P., Khanduja, K.L. and Sharma, R.R. (1986) Effect of cigarette smoke inhalation on certain pulmonary and hepatic drug metabolizing enzymes in rat in vitamin A deficiency. Indian J. Med. Res., 84, 301-309.
