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Effects of β-carotene on chemically-induced skin tumors in HRASkh hairless mice
163
CancerLetters,51(1990)163-168 Elsevier Scientific Publishers Ireland Ltd.
Effects of p-carotene on chemically-induced HRA/Skh hairless mice H.H. Steinel and R.S.U. Toxicology Unit, 2006 (Australia)
Baker
Institute of Occupational Health
National
(Received 5 December 1989) (Revision received 2 February (Accepted 5 February 1990)
In order to evaluate the role ofa-carotene as an inhibitor of skin carcinogenesis, hairless female HRA/Skh mice were treated with the initiator 7,12-dimethylbenz[a]anthracene (DMBA) and with the promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), and were fed a balanced diet free from vitamin A either with or without gavage-administered /3-carotene. There was no evidence of avitaminosis A or differences in body weight in mice deprived of p-carotene and vitamin A, compared with those given 290 or 1430 ZU p-carotene/kg per day. Mice fed with normal animal feed pellets displayed a significantly higher body weight (28.5 f 1.95 g) compared with mice on the special diet (25.7 & 1.9 g), and also displayed a higher papilloma yield. However animals on the special diet, fed with /?-carotene from significantly lower weaning, displayed numbers of papillomas per mouse. This lower papilloma yield was evident particularly between 12-24 weeks after commencement of the study, which coincided with the period of maximum tumor yield in DMBA/TPAtreated mice. The characteristic regression of papillomas after that time points to the reversi-
0304-3835/90/$03.50
Published
to: R.S.U.
Baker.
0
1990 El sevier Scientific Publishers
and Printed in Ireland
and
Safety,
Building A27,
University
of Sydney, N.S. W.
1990)
Summary
Correspondence
skin tumors in
bility of many earlier papillomas, and their dependence on continued TPA administration. Evaluation of carcinomas in mice on the various dietary regimes showed there was no significant difference between any group, including those fed with /I%carotene continuously from weaning. The present results demonstrate that a sustained dietary intake of p-carotene from 3 weeks of age partially suppressed the growth of papillomas, but did not affect the course of malignant progression in DMBA/TPA-treated HRA/Skh mice. It is evident that &carotene predominantly affects TPA-dependent papillomas, which possess reversible properties and have a low probability of progression to form carcinomas.
Keywords: hairless mice; initiation/promotion; skin; tumor suppression; p-carotene. Introduction Epidemiological studies have indicated that cancer patients have lower blood levels of vitamin A 16,121. It has also been shown that decreased consumption of foods containing vitamin A or its dietary precursor, p-carotene, leads to an increased risk of contracting certain cancers [El. In investigations of chemoprevention, retinoids have been shown to pro-
Ireland Ltd
164
tect experimental animals against cancers of the skin [8,18], mammary gland and urinary bladder [ 111. However, not all reports agree. Topical retinoic acid enhanced development of UV-induced skin cancer [4], while vitamin A incorporated in food increased occurrence of gamma-radiation-induced lung adenomas [lo], and failed to suppress either 7,12-dimeinducedthylbenz[a]anthracene (DMBA) mammary cancer [19] or UV-induced skin cancer [5]. Although retinoic acid did not reduce the incidence of DMBA-induced rat salivary gland cancer [ 11, dietary p-carotene afforded some protection. Furthermore, topical B-carotene was reported to inhibit DMBAinduced oral cancer in hamsters [16]. Thus, evidence for protection by vitamin A against experimentally-induced cancers remains equivocal, while it appears possible that the provitamin may itself be protective. However, it has yet to be established whether normal dietary levels of p-carotene have a significant protective role. The present study examines the action of p-carotene on chemical induction of skin cancer and against established tumors. Materials and methods 12-0-Tetradecanoylphorbol-13-acetate 7,12-dimethylbenz[a]anthracene (DMBA) and trans-p-carotene were purchased from Sigma Chemical Company. Acetone (Ajax), ethanol (Merck) and retail grade peanut oil were employed as solvents. Mature inbred female HRA/Skh mice [15] at 42-49 days of age were employed in all experiments. The mice were housed, 10 animals per plastic box (P. McGilI & Co.), on vermiculite bedding (Boral) and maintained at a temperature of 23-25OC with a 12-h day/ night cycle. Lighting was provided by gold lamps (GEC F40GO) which emit minimal UV light. Water and food were freely accessible. Mice were initiated with a single treatment of 2.56 pg (10 nmol) DMBA in 50 ~1 acetone applied over the lower half of their dorsal skin surface. After 1 week, promotion was commenced with 1 pg (1.6 nmol) TPA in 50 ~1
(TPA),
acetone applied twice weekly over the same surface for a further 19 weeks (i.e., promoter treatment ended 20 weeks after initiation). This regime was chosen in order to produce a significant but not excessive number of tumors so that any suppressive effect of p-carotene would not be masked [15]. Groups of 20 female mice were used for each experiment. The tumors, which were usually papillomas, were counted weekly and expressed as the number of tumors per mouse (papilloma yield) or percent of papilloma-bearing animals (incidence). At the conclusion of each experiment carcinoma-like tumors were examined histopathologically. All animals dying before this time were autopsied and suspect tumors analyzed. Although the study period was O-40 weeks, mice were held for a further 12 weeks to allow additional time for the appearance of carcinomas. From 3 weeks of age, mice were provided with a special vitamin A- and /3-carotene-free diet [5] consisting of sucrose (8% w/w), dextrose (27%), corn flour (30%), soya protein (21%), cellulose (5%), peanut oil (6%) and a vitamin-mineral premix (3 %) containing 12 vitamins or cofactors and 8 trace elements conforming with NIH guidelines of recommended minimum essential dietary requirements for rodents [13], except that vitamin A was omitted from the premix. B-Carotene and vitamin A were undetectable by HPLC analysis (G. Kelly, pers. commun.) . One week after initiation, animals were divided into three groups receiving either plain peanut oil or 5 x 10 IU @carotene/week (290 IU/kg per day) or 5 x 50 IU /&carotene/week (1430 IU/kg per day) in 100 ~1 peanut oil, administered by gavage 5 days/week. Results were pooled from two separate experiments. Mice maintained on a normal diet of animal pellets (Allied Feed) were compared with those on the special formulation in a single experiment, In order to investigate the effect of /3-carotene on established tumors, mice on normal feed pellets were initiated and then promoted for 19 weeks, after which they were randomly distributed into two groups. Both received the
165
vitamin A-free diet from week 20-40, one group being gavage-fed 5 x 50 IU /3-carotene/week dissolved in peanut oil, while the other was administered plain peanut oil. The two-tailed unpaired t-test was used to tumor yields between different compare groups, at individual time points.
Results and discussion Mice on the normal diet had significantly higher body weights (28.5 f 1.95 g) after 20 weeks compared with animals maintained on the special diet (P < 0.0001). The latter, whether lacking carotenoid supplement or gavage-fed with 290 or 1430 IU p-carotene/kg per day, had equivalent (P = 0.57) weights (25.7 f 1.9, 25.4 & 1.7 and 25.2 + 1.4g, respectively) which remained lower than those of mice on the normal diet until the conclusion of the experiment. This weight difference was accompanied by significantly lower tumor yields in all groups on the special diet compared with mice on the normal diet (Fig. lA, Table 1)) particularly between weeks 12-18 (P< 0.01 at each week). This is in accord with the known relationship between tumor occurrence and body weight in mice [20]. 6
TPA
promotion
Cessation of TPA promotion after 20 weeks resulted in an expected downturn in papilloma numbers [ 151 in all treatment groups (Fig. 1A). In mice maintained on the vitamin A-lacking special diet, oral administration of p-carotene further reduced the papilloma yield and incidence without having any effect on body weight. The effect of j&carotene was evident particularly between weeks 12 and 20 (P < 0.01 at each week) i.e., before completion of TPA treatments. Papilloma yields were reduced by 44% and 46% at week 20, in mice given 290 IU or 1430 IU p-carotene/kg per day, respectively, without any obvious doseresponse. In the final weeks of the experiment, the differences in papilloma yield between the different groups became less apparent (Fig. lA, Table l), although the incidence of papilloma-bearing animals remained lower in ficarotene-fed mice on the special diet (Table 1). The yield of carcinomas at week 52 did not differ statistically between any of the four groups. When 1430 IU B-carotene/kg per day was fed to mice with already well-developed papillomas, commencing at week 20, there was no significant reduction in tumor yield compared with mice on the retinoid and carotenoid-free diet (Fig. 1B). Nor was there any significant difference in carcinoma occurrence in the two
6
(XIX)
I
TPA
promot,on
(38x)
B
4 *-
Papillomad mouse
Papillomas/ mouse
D
10
20
30
40
Time (weeks)
Fig. 1.
(A) Skin papilloma yield of initiated and promoted HRA/Skh mice fed on normal mouse feed pellets (0)) or on a special vitamin A-lacking dietary formulation gavage-fed with peanut oil (0) with 290 IU fLcarotene/kg per day (+) or with 1430 IU p-carotene/kg per day (H) h om weaning. (B) Skin papilloma yield of initiated and promoted HRA/Skh mice changed to a special vitamin A-lacking diet and gavage-fed with peanut oil (0) or 1430 IU o-carotene/kg per day (a) at week 20.
20 40
Normal Specialb
0.17
0.17
40
40
0.32 0.22
2.5 f 2.4’
2.5 + 2.1c
4.9 & 1.8 3.5 f 1.6
Maximum paps/mouse R f S.D.
HRA/Skh
77
77
100 97
(58)
Papilloma incidence at 20 weeks
in DMBA- and TPA-treated
- 0.05
- 0.07
- 0.15 -0.11
(paps/w)
Papilloma decrease over 18-30 weeks
mice maintained
1.2 f
1.7
0.8 & 1.0
1.6 f 1.2 1.2 + 1.3
Minimum paps/mouse x f S.D.
52
50
78 68
(%)
Papilloma incidence at 40 weeks
on different diets from weaning.
8/37
7/38
5/19 7/25
Carcinomas at 52 weeksa (no./no. of surviving mice)
“Normal diet, 5 SCC; special diet, 6 SCC, 1 CiS; 290 III/kg/day, 4 SCC, 3 CiS; 1430 W/kg/day, 8 SCC. CiS usually progress into SCC. SCC, squamous cell carcinomas; CiS, carcinomas in situ. bLoss of 10 mice due to dehydration. Significantly different from control animals fed normal or unsupplemented special diets (P < 0.05). Tumor yields in control mice on the special diet were also significantly different from those on the normal diet (P < 0.05).
Special + p-carotene (290 Ill/kg/day) Special + p-carotene (1430 RI/kg/day)
Papilloma increase over 6- 14 weeks
No. of mice
Diet
(paps/w)
occurrence
Papilloma
and carcinoma
Table 1.
167
groups. Mice without p-carotene developed 3 squamous cell carcinomas (SCC) amongst 19 animals. Those fed p-carotene had 6 SCC amongst 16 surviving animals. Thus, effects of p-carotene required continuous feeding from weaning age onwards. Although the approach used in this study does not distinguish the initiation from the promotion phase, many of the papillomas suppressed by p-carotene were promoter-dependent [2] since (i) the increase in papilloma numbers between weeks 6-14 and their decrease between weeks 18-30 were smaller in /3-carotene-fed mice (Table 1); (ii) TPA-independent papillomas, with a high probability of progressing into carcinomas [2], were little affected and (iii) permanent papillomas, remaining after 30 weeks, were unaffected by p-carotene feeding. Although other reports describe similar effects, our results show that p-carotene is able to reduce papilloma occurrence at carotenoid levels considerably lower than those employed previously. The lowest effective p-carotene dose in this study was 290 IU/kg per day compared with WHO-recommended dietary intake levels of 115 IU/kg per day of vitamin A studies have (i.e. p-carotene). Other demonstrated tumor reductions after injections of 120,000 IU/kg per day [3], after feeding 9,200,OOO IU/kg per day [9], and after topical application of 4400 IU/kg per day [ 161. The lack of a dose response in this study indicates that p-carotene doses administered here may be close to the absorption limits for HRA/Skh mice. B-Carotene is converted in the intestine to vitamin A aldehyde, the intermediate product in vitamin A metabolism. However, some p-carotene will pass unaltered into the body [7]. It is known that feeding large doses of p-carotene results in significant levels of both the carotenoid and vitamin A in the skin [9]. While retinoic acid is known to act as an antipromoter in mouse skin carcinogenesis 114,171, the present results cannot exclude the possibility that p-carotene itself possesses some intrinsic antipromotional activity, without necessarily undergoing conversion to vitamin A.
Acknowledgements The authors are grateful for assistance from Dr P. Canfield and Mr. G. Greenoak, Department of Veterinary Pathology, University of Sydney. Thanks are also due to Professor A.W. Murray, School of Biology, Flinders University, South Australia, and to Dr A.M. Bonin for helpful advice during this research. This study was supported by a grant from the N.S.W. Cancer Council. References 1
2
3
4
5
6
7
8
9
10
11
12
Alam, B.S., Alam, S.Q., Weir, J.C., Jr. and Gibson, W.A. (1984) Chemopreventive effects of fl-carotene and Id-cis-retinoic acid on salivary gland tumors. Nutr. Cancer, 6,4-12. Burns, F.J., Vanderlan M., Snyder, E. and Albert, R.E. (1978) Induction and progression kinetics of mouse skin papillomas. In: Mechanism of Tumor Promotion and Cocarcinogenesis, Vol. 2, pp. 91-96. Editors: T.J. Slaga, A. Sivak and R.K. Boutwell. Raven Press, New York. Epstein, J.H. (1977) Effects of p-carotene on ultraviolet induced cancer formation in the hairless mouse skin. Photochem. Photobiol., 25, 211-213. Forbes, P.D., Urbach, F. and Davies, R.E. (1987) Enhancement of experimental photocarcinogenesis by topical retinoic acid. Cancer Lett., 7, 85-90. Kelly, G.E., Meikle, W.D. and Sheil, A.G.R. (1989) Effects of oral retinoid (vitamin A and etretinate) therapy on photocarcinogenesis in hairless mice. Photochem. Photobiol., 50, 213-215. Kummet, T. and Meyskens, potential inhibitor of human 281-289.
F.L. (1983) Vitamin A: A cancer. Semin. Oncol., 10,
Lentner, C. (1986) Metabolism of vitamin A. In: Geigy Scientific Tables, Vol. 4 n. 147, Ciba Geigy Limited, Basle, Switzerland. Matter, A. and Bollag, W. (1977) A fine structural study on the therapeutic effect of an aromatic retinoid on chemically-induced skin papili- 7-e of the mouse. Eur. J. Cancer, 13,831-838. Mathews-Roth, M.M. (1982) Antirumor activity of betacarotene, canthaxanthin and nhytoene. Oncology, 39, 33 -37. Mian, T.A., Theiss, J.C. and Geseii T.F. (1984) Effect of vitamin A on lung tumorigenesis ill ...adiated and unirradiated strain A mice. Cancer Lett., ZL, 103- 112. Moon, R.C., McCormick, D.L. and Metha. R.G. (1983) inhibition of carcinogenesis by retinoids. Cancer Res., 43, 2469s-2475s. Peto, R., Doll, R., Buckley, J.D. and Sporn, M.B. (1981) Can dietary beta-carotene materially reduce human cancer rates? Nature, 290, 201-208.
168
13
14
15
16
17
Report
of the American
Institute of Nutrition
and Ad Hoc
Committee on Standards for Nutrition Studies (1977) J. Nutr., 107, 1340-1348. Slaga, T.J., Klein-Szanto, A.J.P., Fisher, S.M., Weeks, C.E., Nelson, K. and Major, S. (1980) Studies on mechanism of action of anti-promoting agents: Their specificity in two-stage promotion. Proc. Natl. Acad. Sci., 77, 22512254. Steinel, H.H. and Baker, R.S.U. (1988) Sensitivity of HRA/Skh mice to initiation promotion of skin tumours by chemical treatment. Cancer Lett., 46,61-65. Suds, D., Schwartz, J. and Shklar, G. (1986) inhibition of experimental oral carcinogenesis by topical beta carotene. Carcinogenesis, 7, 711-715. Verma, A.K. (1987) Inhibition of both Stage I and Stage II mouse skin tumor promotion by retinoic acid and the
dependence
18
19
20
of inhibition of tumor promotion
on the dura-
tion of retinoic acid treatment. Cancer Res., 47, 50975101. Weeks, C.E., Slaga, T.J., Hennings, H., Gleason, G.L. and Bracken, W.M. (1979) Inhibition of phorbol esterinduced tumor promotion in mice by vitamin A analog and anti-inflammatory steroid. J. Natl. Cancer Inst., 63, 401406. Welsch, C.W., DeHoog, J.V. and Moon, R.C. (1984) Lack of an effect of dietary retinoids in chemical carcinogenesis of the mouse mammary gland: inverse relationship between mammary tumour cell anaplasia and retinoid efficacy. Carcinogenesis, 5, 1301-1304. White, R.F. (1961) The relationship between underfeeding and tumor formation, transplantation, and growth in rats and mice. Cancer Res., 21,281-290.
CancerLetters,51(1990)163-168 Elsevier Scientific Publishers Ireland Ltd.
Effects of p-carotene on chemically-induced HRA/Skh hairless mice H.H. Steinel and R.S.U. Toxicology Unit, 2006 (Australia)
Baker
Institute of Occupational Health
National
(Received 5 December 1989) (Revision received 2 February (Accepted 5 February 1990)
In order to evaluate the role ofa-carotene as an inhibitor of skin carcinogenesis, hairless female HRA/Skh mice were treated with the initiator 7,12-dimethylbenz[a]anthracene (DMBA) and with the promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), and were fed a balanced diet free from vitamin A either with or without gavage-administered /3-carotene. There was no evidence of avitaminosis A or differences in body weight in mice deprived of p-carotene and vitamin A, compared with those given 290 or 1430 ZU p-carotene/kg per day. Mice fed with normal animal feed pellets displayed a significantly higher body weight (28.5 f 1.95 g) compared with mice on the special diet (25.7 & 1.9 g), and also displayed a higher papilloma yield. However animals on the special diet, fed with /?-carotene from significantly lower weaning, displayed numbers of papillomas per mouse. This lower papilloma yield was evident particularly between 12-24 weeks after commencement of the study, which coincided with the period of maximum tumor yield in DMBA/TPAtreated mice. The characteristic regression of papillomas after that time points to the reversi-
0304-3835/90/$03.50
Published
to: R.S.U.
Baker.
0
1990 El sevier Scientific Publishers
and Printed in Ireland
and
Safety,
Building A27,
University
of Sydney, N.S. W.
1990)
Summary
Correspondence
skin tumors in
bility of many earlier papillomas, and their dependence on continued TPA administration. Evaluation of carcinomas in mice on the various dietary regimes showed there was no significant difference between any group, including those fed with /I%carotene continuously from weaning. The present results demonstrate that a sustained dietary intake of p-carotene from 3 weeks of age partially suppressed the growth of papillomas, but did not affect the course of malignant progression in DMBA/TPA-treated HRA/Skh mice. It is evident that &carotene predominantly affects TPA-dependent papillomas, which possess reversible properties and have a low probability of progression to form carcinomas.
Keywords: hairless mice; initiation/promotion; skin; tumor suppression; p-carotene. Introduction Epidemiological studies have indicated that cancer patients have lower blood levels of vitamin A 16,121. It has also been shown that decreased consumption of foods containing vitamin A or its dietary precursor, p-carotene, leads to an increased risk of contracting certain cancers [El. In investigations of chemoprevention, retinoids have been shown to pro-
Ireland Ltd
164
tect experimental animals against cancers of the skin [8,18], mammary gland and urinary bladder [ 111. However, not all reports agree. Topical retinoic acid enhanced development of UV-induced skin cancer [4], while vitamin A incorporated in food increased occurrence of gamma-radiation-induced lung adenomas [lo], and failed to suppress either 7,12-dimeinducedthylbenz[a]anthracene (DMBA) mammary cancer [19] or UV-induced skin cancer [5]. Although retinoic acid did not reduce the incidence of DMBA-induced rat salivary gland cancer [ 11, dietary p-carotene afforded some protection. Furthermore, topical B-carotene was reported to inhibit DMBAinduced oral cancer in hamsters [16]. Thus, evidence for protection by vitamin A against experimentally-induced cancers remains equivocal, while it appears possible that the provitamin may itself be protective. However, it has yet to be established whether normal dietary levels of p-carotene have a significant protective role. The present study examines the action of p-carotene on chemical induction of skin cancer and against established tumors. Materials and methods 12-0-Tetradecanoylphorbol-13-acetate 7,12-dimethylbenz[a]anthracene (DMBA) and trans-p-carotene were purchased from Sigma Chemical Company. Acetone (Ajax), ethanol (Merck) and retail grade peanut oil were employed as solvents. Mature inbred female HRA/Skh mice [15] at 42-49 days of age were employed in all experiments. The mice were housed, 10 animals per plastic box (P. McGilI & Co.), on vermiculite bedding (Boral) and maintained at a temperature of 23-25OC with a 12-h day/ night cycle. Lighting was provided by gold lamps (GEC F40GO) which emit minimal UV light. Water and food were freely accessible. Mice were initiated with a single treatment of 2.56 pg (10 nmol) DMBA in 50 ~1 acetone applied over the lower half of their dorsal skin surface. After 1 week, promotion was commenced with 1 pg (1.6 nmol) TPA in 50 ~1
(TPA),
acetone applied twice weekly over the same surface for a further 19 weeks (i.e., promoter treatment ended 20 weeks after initiation). This regime was chosen in order to produce a significant but not excessive number of tumors so that any suppressive effect of p-carotene would not be masked [15]. Groups of 20 female mice were used for each experiment. The tumors, which were usually papillomas, were counted weekly and expressed as the number of tumors per mouse (papilloma yield) or percent of papilloma-bearing animals (incidence). At the conclusion of each experiment carcinoma-like tumors were examined histopathologically. All animals dying before this time were autopsied and suspect tumors analyzed. Although the study period was O-40 weeks, mice were held for a further 12 weeks to allow additional time for the appearance of carcinomas. From 3 weeks of age, mice were provided with a special vitamin A- and /3-carotene-free diet [5] consisting of sucrose (8% w/w), dextrose (27%), corn flour (30%), soya protein (21%), cellulose (5%), peanut oil (6%) and a vitamin-mineral premix (3 %) containing 12 vitamins or cofactors and 8 trace elements conforming with NIH guidelines of recommended minimum essential dietary requirements for rodents [13], except that vitamin A was omitted from the premix. B-Carotene and vitamin A were undetectable by HPLC analysis (G. Kelly, pers. commun.) . One week after initiation, animals were divided into three groups receiving either plain peanut oil or 5 x 10 IU @carotene/week (290 IU/kg per day) or 5 x 50 IU /&carotene/week (1430 IU/kg per day) in 100 ~1 peanut oil, administered by gavage 5 days/week. Results were pooled from two separate experiments. Mice maintained on a normal diet of animal pellets (Allied Feed) were compared with those on the special formulation in a single experiment, In order to investigate the effect of /3-carotene on established tumors, mice on normal feed pellets were initiated and then promoted for 19 weeks, after which they were randomly distributed into two groups. Both received the
165
vitamin A-free diet from week 20-40, one group being gavage-fed 5 x 50 IU /3-carotene/week dissolved in peanut oil, while the other was administered plain peanut oil. The two-tailed unpaired t-test was used to tumor yields between different compare groups, at individual time points.
Results and discussion Mice on the normal diet had significantly higher body weights (28.5 f 1.95 g) after 20 weeks compared with animals maintained on the special diet (P < 0.0001). The latter, whether lacking carotenoid supplement or gavage-fed with 290 or 1430 IU p-carotene/kg per day, had equivalent (P = 0.57) weights (25.7 f 1.9, 25.4 & 1.7 and 25.2 + 1.4g, respectively) which remained lower than those of mice on the normal diet until the conclusion of the experiment. This weight difference was accompanied by significantly lower tumor yields in all groups on the special diet compared with mice on the normal diet (Fig. lA, Table 1)) particularly between weeks 12-18 (P< 0.01 at each week). This is in accord with the known relationship between tumor occurrence and body weight in mice [20]. 6
TPA
promotion
Cessation of TPA promotion after 20 weeks resulted in an expected downturn in papilloma numbers [ 151 in all treatment groups (Fig. 1A). In mice maintained on the vitamin A-lacking special diet, oral administration of p-carotene further reduced the papilloma yield and incidence without having any effect on body weight. The effect of j&carotene was evident particularly between weeks 12 and 20 (P < 0.01 at each week) i.e., before completion of TPA treatments. Papilloma yields were reduced by 44% and 46% at week 20, in mice given 290 IU or 1430 IU p-carotene/kg per day, respectively, without any obvious doseresponse. In the final weeks of the experiment, the differences in papilloma yield between the different groups became less apparent (Fig. lA, Table l), although the incidence of papilloma-bearing animals remained lower in ficarotene-fed mice on the special diet (Table 1). The yield of carcinomas at week 52 did not differ statistically between any of the four groups. When 1430 IU B-carotene/kg per day was fed to mice with already well-developed papillomas, commencing at week 20, there was no significant reduction in tumor yield compared with mice on the retinoid and carotenoid-free diet (Fig. 1B). Nor was there any significant difference in carcinoma occurrence in the two
6
(XIX)
I
TPA
promot,on
(38x)
B
4 *-
Papillomad mouse
Papillomas/ mouse
D
10
20
30
40
Time (weeks)
Fig. 1.
(A) Skin papilloma yield of initiated and promoted HRA/Skh mice fed on normal mouse feed pellets (0)) or on a special vitamin A-lacking dietary formulation gavage-fed with peanut oil (0) with 290 IU fLcarotene/kg per day (+) or with 1430 IU p-carotene/kg per day (H) h om weaning. (B) Skin papilloma yield of initiated and promoted HRA/Skh mice changed to a special vitamin A-lacking diet and gavage-fed with peanut oil (0) or 1430 IU o-carotene/kg per day (a) at week 20.
20 40
Normal Specialb
0.17
0.17
40
40
0.32 0.22
2.5 f 2.4’
2.5 + 2.1c
4.9 & 1.8 3.5 f 1.6
Maximum paps/mouse R f S.D.
HRA/Skh
77
77
100 97
(58)
Papilloma incidence at 20 weeks
in DMBA- and TPA-treated
- 0.05
- 0.07
- 0.15 -0.11
(paps/w)
Papilloma decrease over 18-30 weeks
mice maintained
1.2 f
1.7
0.8 & 1.0
1.6 f 1.2 1.2 + 1.3
Minimum paps/mouse x f S.D.
52
50
78 68
(%)
Papilloma incidence at 40 weeks
on different diets from weaning.
8/37
7/38
5/19 7/25
Carcinomas at 52 weeksa (no./no. of surviving mice)
“Normal diet, 5 SCC; special diet, 6 SCC, 1 CiS; 290 III/kg/day, 4 SCC, 3 CiS; 1430 W/kg/day, 8 SCC. CiS usually progress into SCC. SCC, squamous cell carcinomas; CiS, carcinomas in situ. bLoss of 10 mice due to dehydration. Significantly different from control animals fed normal or unsupplemented special diets (P < 0.05). Tumor yields in control mice on the special diet were also significantly different from those on the normal diet (P < 0.05).
Special + p-carotene (290 Ill/kg/day) Special + p-carotene (1430 RI/kg/day)
Papilloma increase over 6- 14 weeks
No. of mice
Diet
(paps/w)
occurrence
Papilloma
and carcinoma
Table 1.
167
groups. Mice without p-carotene developed 3 squamous cell carcinomas (SCC) amongst 19 animals. Those fed p-carotene had 6 SCC amongst 16 surviving animals. Thus, effects of p-carotene required continuous feeding from weaning age onwards. Although the approach used in this study does not distinguish the initiation from the promotion phase, many of the papillomas suppressed by p-carotene were promoter-dependent [2] since (i) the increase in papilloma numbers between weeks 6-14 and their decrease between weeks 18-30 were smaller in /3-carotene-fed mice (Table 1); (ii) TPA-independent papillomas, with a high probability of progressing into carcinomas [2], were little affected and (iii) permanent papillomas, remaining after 30 weeks, were unaffected by p-carotene feeding. Although other reports describe similar effects, our results show that p-carotene is able to reduce papilloma occurrence at carotenoid levels considerably lower than those employed previously. The lowest effective p-carotene dose in this study was 290 IU/kg per day compared with WHO-recommended dietary intake levels of 115 IU/kg per day of vitamin A studies have (i.e. p-carotene). Other demonstrated tumor reductions after injections of 120,000 IU/kg per day [3], after feeding 9,200,OOO IU/kg per day [9], and after topical application of 4400 IU/kg per day [ 161. The lack of a dose response in this study indicates that p-carotene doses administered here may be close to the absorption limits for HRA/Skh mice. B-Carotene is converted in the intestine to vitamin A aldehyde, the intermediate product in vitamin A metabolism. However, some p-carotene will pass unaltered into the body [7]. It is known that feeding large doses of p-carotene results in significant levels of both the carotenoid and vitamin A in the skin [9]. While retinoic acid is known to act as an antipromoter in mouse skin carcinogenesis 114,171, the present results cannot exclude the possibility that p-carotene itself possesses some intrinsic antipromotional activity, without necessarily undergoing conversion to vitamin A.
Acknowledgements The authors are grateful for assistance from Dr P. Canfield and Mr. G. Greenoak, Department of Veterinary Pathology, University of Sydney. Thanks are also due to Professor A.W. Murray, School of Biology, Flinders University, South Australia, and to Dr A.M. Bonin for helpful advice during this research. This study was supported by a grant from the N.S.W. Cancer Council. References 1
2
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