145
Cancer Letters. 57 (1991) 145- 152 Elsevier Scientific Publishers Ireland Ltd.
Effect of dietary calorie and fat restriction on mammary tumor growth and hepatic as well as tumor glutathione in rats P. Zhu, E. Frei, B. Bunk, Institute of Toxicology Heidelberg
M.R. Berger and D. Schm8hla
and Chemotherapy,
German
Cancer
(Received
5 February
1991)
(Accepted
5 February
1991)
Summary Effect of dietary calorie restriction and fat reduction on growth of established mammary carcinoma in rats and on glutathione levels in liuer and tumor tissue was investigated. Reduced (GSH) and oxidized (GSSG) glutathione were determined enzymatically. Female Sprague-Dawley rats were injected with 25 mg/kg methylnitrosourea (MNU) on day 50 of life for tumor induction, and subsequently fed a diet containing 50 kcal/day with 45% (energy 48) fat. When tumors reached approximately 1 cm3, the diet was changed for 10 A 2 weeks.
Four dietary groups were formed: two high calorie groups (50 kcal/day) with 45% or 25 !%Ifat and two calorie restricted groups (35 kcal/day) with 45% or 25% fat, respectively. Tumor growth was significantly inhibted by the 30 % calorie restriction, and the inhibition
restricted reduction
was most effective in the calorie group with low fat level. However, of fat, alone, had no significant in-
hibitory effect. GSSG levels in both liver and tumor showed no differences among the groups. Hepatic GSH levels tended to be lower showed
Research
Center,
groups
with
Im Neuenheimer
Feld
280.
D-6900
(F.R.G.)
in the no
calorie-restricted difference
groups,
between
Correspondence to: M.R. Berger, Institute of Toxicology German Cancer Research Center, Chemotherapy, Neuenheimer Feld 280, D-6900 Heidelberg, F.R.G.
0304-3835/91/$03.50 Published and Printed
and
isocaloric and Im
0 1991 El sevier Scientific Publishers in Ireland
different
fat
levels.
In
contrast,
GSH in tumor tissue tended to be lower in the low fat groups, independently of caiorie levels.
Keywords: glutathione; mammary carcinoma
dietary
restriction;
Introduction The effect of different dietary calorie levels and/or fat intake on mammary tumorigenesis in rats has been intensively investigated [4,6-8,16,18,19,29,30,32,35]. Freedman et al. [12] analysed data from 100 animal experiments and reported that both high calorie and high fat intake independently increased mammary tumor incidence, and that the effect of fat was only 2/3 of the calorie effect both in SpragueDawley rats and in mice. Reviews of animal studies on the relationship between fat intake and mammary tumor incidence by other authors yielded inconsistent and inconclusive results [3,28]. The mechanisms by which dietary restriction prolongs life and inhibits age-associated diseases [33], including many cancers [l], are still not clear. Dietary restriction could be involved in the processes regulating cellular detoxification and redox systems against oxidative damage. It has been reported that diet can change the GSH level and the activity of Ireland Ltd.
146
GSH-related enzymes in liver and other tissues [ 11,2 11. Correlations between GSH and/or GSH-related enzymes and carcinogenesis have been reported in many studies [10,14,17,23,25]. GSH is co-substrate in the GSH-peroxidase reaction for the reduction of hydroperoxides in the defense against oxidative stress [27]. Hence, an alteration in the glutathione redox status could have an influence on the tumor growth, and/or, indicate metabolic disturbances contributing to tumor progression. A life-long dietary restriction as a method for prevention of cancer is not very realistic in the human situation. We therefore investigated the influence of calorie restriction and fat content, on the growth of methylnitrosourea (MNU)-induced mammary tumors after their manifestation. The status of glutathione in the liver and in the mammary tumors, and its possible correlation with tumor growth in the different calorie and fat groups were also studied. Materials and Methods Animals Female Sprague-Dawley rats were purchased from the Zentalinstitut ftir Versuchstierzucht, Hannover, F.R.G., at the age of 40 days. The whole experiment lasted 30 weeks. Chemicals
and reagents
MNU was synthesized in our institute (M. Na2HP04, Na,EDTA, Wiessler) . NaH2P04, 5-sulfosalicylic acid, 2-vinylpyridine stabilized with 0.1% 4-tert-butylbenzcatechin, triethanolamine and Tris(hydroxymethyl)-aminomethan were supplied by Merck, Darmstadt, F.R.G. 5,5’-Dithio-bis(-2-nitrobenzoic acid) (DTNB, Ellman’s reagent), GSH, GSSG, NADPH and glutathione reductase (600 (98%) units/ml) were purchased from Boehringer Mannheim GmbH, Mannheim, F.R.G. Bovine serum albumin (BSA) was supplied by Serva, Heidelberg, F.R.G. Bio-Rad Protein Assay Kit was purchased by Bio-Rad Laboratories GmbH, Munich, F.R.G.
Mammary
tumor
induction
Rats (116) were injected via the tail vein with 25 mg/kg MNU on day 50 of life to induce mammary carcinoma. Diet and feeding
Each rat was placed in an individual cage in a temperature (21 f Z’C), light (12-h light, 12-h dark) and humidity (60%) controlled room. The animals were given tap water ad libitum. Prior to tumor induction, animals were fed a standard commercial diet (Altromin 1320, Lage, F.R.G.). One day after MNU injection, all the rats were fet a diet containing 50 kcal/day and 45% (energy %a) fat. When tumors reached approximately 1 cm3, animals were randomized into the following four dietary groups for 10 f 2 weeks: group I: 50 kcal/day 45% fat (high calorie and high fat); group 11: 35 kcal/day 45% fat (calorie restricted); group III: 50 kcal/day 25% fat (high calorie and fat reduced); group IV: 35 kcal/day 25% fat (calorie restricted and fat reduced). The composition of diets is shown in Table I. All diets were obtained from Unilever Research, Rotterdam, Netherlands, kept at - 20°C and thawed before use. Rats were fed every morning and weighed and palpated for tumors weekly. Liver
and tumor
preparation
At the end of the experiment, rats were exsanguinated under ether anesthesia and a fraction of liver tissue was removed after perfusion with 0.9% NaCl. All tumors were excised, weighed and cleaned from connective and necrotic tissue. Parts of the tumors were fixed in 10% formalin for histological examination (D. Komitowski and F. Amelung, Institute of Experimental Pathology, German Cancer Heidelberg, F.R.G.). Research Center, Prepared remaining tissues were immediately frozen in liquid nitrogen and stored at - 70°C until assayed. All the samples were collected at 1200 h to minimize a possible diurnal fluctuation of glutathione.
147 Table
1.
Dietary
groups
and
composition
ND25”
ND45”
Diet kcal/day (Group
(l) 35
no.)
Composition:
g/100
of diets.
(II)
50
(III) 35
g
27.80
23.90
Corn
starch
41.00
57.40
Palm
oil
16.28
7.80
3.04
1.46
Casein
Lard
2.39
1.14
Cellulose
6.70
5.80
Vitamins
0.45
0.39
Minerals
2.42
2.08
24.00
24.00
31.00
51.00
45.00
25.00
15.00
15.00
Sunflower
Protein
(%)h
(%)
Carbohydrate Fat Fiber
oil
seed
(W) (mg/kcal)
Vitamins
(mg/kcal)
1.00
1.00
Minerals
(mg/kcal)
5.40
5.40
“ND = normal for group diets
West
German
bPercentage
diet; ND45
for group
III and IV; the fat composition
thetic
were
(IV)
according
to the
I and II. and ND25 of these
fat profile
semisyn-
of ‘normal’
diet. of total
specificity. Briefly, every test cuvette contained 700 ~1 NADPH (0.3mM) in phosphate buffer phosphate, 6.3 mM (143 mM sodium Na2EDTA, pH 7.5), 100 ~1 DTNB (6 mM) in phosphate buffer, 175 ~1 HP0 and 25 ~1 sample. Change in absorption was determined following addition of 10 ~1 glutathione reductase (266 U/ml in phosphate buffer) in triplicate using a Perkin-Elmer Lambda 5 UV/VIS Photometer at 412 nm. The reference cuvette contained 25 ~1 5% 5-sulfosalicylic acid instead of sample. To determine GSSG, GSH was derivatized with 2-vinylpyridine. Triethanolamine (1~2 (w/w) diluted with H20) was added so that the pH was between 6 and 7. After 60 min reaction, GSSG determination was carried out with DTNB-GSSG reductase recycling assay as described above. Standard samples of GSH and GSSG were treated in the same way, and the amount of GSH and GSSG was calculated from the standard curve.
energy.
Determination of GSH and GSSG Liver and tumor tissues from some rats (as indicated in Tables III and IV) were powdered frozen in nitrogen with a Micro-Dismembrator (B. Braun Melsungen AG, F.R.G.) and homogenized in 5% 5-sulfosalicylic acid to precipitate protein. The homogenates were centrifuged at 4000 rev./min for 6 min at 4°C. The supernatant was used for total glutathione (GSH + GSSG, in GSH equivalents) and GSSG determination. GSH and GSSG were determined with a DNTB-GSSG reductase recycling assay according to Griffith and Anderson [2,13] with minor modifications. One fraction of liver and l-2 tumor samples per rat were assayed. All samples were diluted to 3 different concentrations between 20- 120 PM for GSH and between 0.5-8.0 PM for GSSG with 5% sulfosalicylic acid. In this range, the assay offers high sensitivity and
Determination of protein content The 5-sulfosalicylic acid precipitates were dissolved with 0.1 M NaOH at 60°C. Protein content was determined after dilution in Tris-HC1 buffer (pH 7.4) with Bio-Rad Protein Assay Kit spetrophotometrically at 595 nm. BSA was used as standard in this assay. Statistical analysis All the data were processed in the Adam system (software for statistical analysis of biomedical data, German Cancer Research Center, Heidelberg, F.R.G.). Comparisons of parameters for statistically significant differences between groups were made by the Wilcoxon Rank Sum test [15]. The mortality within the groups was compared by Fisher’s exact test [24]. Results Influence
of calorie and fat restriction
on tumor
growth
At the time of tumor manifestation
(18 * 4
148
weeks after MNU injection) 90 rats (78% of MNU injected rats) bearing a tumor volume of 1 cm3 were randomized into the 4 dietary groups. Histology of the tumors revealed 94% adenocarcinoma and 6 % adenoma. The comparison of dietary groups I and II, and groups III and IV, at 10 f 2 weeks after dietary showed that body weight, tumor change, number and tumor weight (Table II) were significantly lower in the calorie restricted groups than in the high calorie groups (P < 0.05). The difference in the ratio of tumor to body weight was still significant (P < 0.05). This indicated that the body weight was less affected by calorie restriction than the tumor weight. The lowest tumor burden was found in group IV, in which rats received low calorie diet with low fat level. Rats in the calorie restricted groups had no loss of body weight during the experiment, but a reduced body weight gain. However, isocaloric diets with reduced fat levels had no significant effect on body weight, tumor number and weight. Mortality was related neither to calorie nor to fat levels. Glutathione status Glutathione in
her Liver weight was significantly reduced by calorie restriction, but showed no differences between the 45% and 25% fat isocaloric groups (Table III). No significant differences in hepatic protein con-
Table II. Diet kcal/day
Effect of dietary calorie content
on tumor growth,
tent per g wet liver tissue among the four dietary groups were observed, indicating that reduction of fat level had no influence on liver protein content. No significant differences were found in GSSG levels. The mean GSH levels were lower in the low calorie groups, although the difference was significant only between groups III and IV. The ratio of GSSG to total glutathione in % (GSSG W) was accordingly in the low calorie groups higher than in the high calorie groups for both fat levels. Furthermore, the GSSG% in the liver was found to be inversely related to tumor weight in group I, when linear regression was used for establishment of the correlation (Fig. 1). In the other three groups coefficient of linear regression (r) was also negative, but did not reach significance. Using all data regardless of diet, the correlation was still significant (r = - 0.44). No correlation between hepatic GSH or GSSG and tumor burden of animals was detected. Glutathione
in tumor
tissue
Table IV shows the glutathione status in mammary tumor tissue. Similar to liver, no significant differences were observed in either tumor protein or GSSG levels between the groups. The GSH levels were significantly reduced in groups III and IV compared to group II (P < 0.05). As a result, the GSSG% in group IV was higher than in group II. A
liver and body weight of rats”
ND45
ND25
(Group no.)
Number of rats Tumor number/rat Tumor weight/rat (g) Body weight (g) Tumor/body weight (W) Mortality ( W)
500)
35(H)
50(111)
35(IV)
21
23
20
2.43 zt 1.12 10.87 +z 7.02 292 zt 29 3.8 zt 2.5 3/24
1.74 l 1.10 6.07 * 7.42 277 f 20 2.2 zt 2.7 l/24
2.35 zt 1.02 7.36 zt 7.03 294 f 15 2.5 +z 2.4 o/20
19 0.95 l 0.71 3.22 zt 4.10 268 zt 23 1.3 f 1.7 3/22
The results are expressed as mean f S.D. aAll parameters except mortality were significantly (P < 0.05, Wilcoxon Rank Sum test).
different
between
group
I and II and between
group
111and IV
149
Table 111. Effect of dietary calorie level on glutathione Diet kcal/day
and protein
content
ND25
ND45 (Group no.)
35(11)
50(I) Number of rats Liver weight/rat (g) Protein mg/g liver GSSG nmol/g liver GSH pmol/g liver GSSG(%)”
16 7.68 107.5 48.71 2.82 1.68
in rat liver.
19 f 0.80 zt 14.72 f 20.00 A 0.70b ztz 0.71d
6.29 103.00 48.52 2.49 1.99
The results are expressed as mean + S.D. “GSSG/(GSH + GSSG) in 46. bcP = 0.016; low calorie groups versus high calorie groups, d.eP = 0.010; low calorie groups versus high calorie groups,
similar, but insignificant difference was also found between groups 1 and III. No correlation between GSSG or GSSG% and tumor weight could be observed. Mean GSH levels in tumor tissue were approximately l/3 (26-38%) of those found in liver, whereas GSSG levels ranged between 56 and 70% of those found in liver, and consequently the GSSG% in tumor was approximately Z-fold higher than in liver.
35(IV)
5O(II!)
+z f zt f f
18 7.58 98.44 50.03 2.89 1.74
0.99 13.91 12.3 0.67c 0.51’
according according
to Wilcoxon to Wilcoxon
18 f 1.28 + 10.24 zt 21.51 A 0.87b A 0.60d
5.73 f 0.51 101.78 f 17.27 51.92 +z 22.35 2.31 ztz 0.66 2.19 f 0.73’
Rank Sum test. Rank Sum test.
In this study we used semisynthetic diets in which fatty acid composition reflected the fatty acid profile of normal West German diet, and
found that a 30% calorie restriction could inhibit the growth of MNU-induced mammary carcinoma in rats, even after tumor manifestation. The most effective inhibition occurred in the low calorie group with low fat level. However, the reduction of fat with complementary increase of carbohydrates and no calorie restriction had no significant inhibitory effect. Reduced weight gain but no weight loss was observed in calorie restricted groups, indicating that mild calorie restriction was used in this study. The development of body weight and tumor growth during the experiment will be described in more detail elsewhere (Bunk et al. in preparation).
Table IV. Effect of dietary fat level on glutathione
in mammary
Discussion
Diet kcal/day
content
ND45
tumors. ND25
(Group no.)
Number of rats Protein mg/kg tumor GSSG nmol/g tumor GSH pmol/g tumor GSSGBc
50(I)
35(1I)
11 73.63 zt 11.11 32.58 ziz 21.16 0.91 f 0.29’ 3.58 f 2.21d
8 68.84 35.07 0.95 3.46
The results are expressed as mean f S.D “.bP = 0.04; low calorie groups versus high calorie groups, ‘GSSG/(GSH + GSSG) W. d.eP c 0.03; low calorie groups versus high calorie groups,
5@(M)
ziz 14.69 f 18.15 f 0.22 ziz 1.22d
5 67.85 28.44 0.74 4.10
35(IV)
f zt + f
7.09 7.19 0.23b 1.74’
according
to Wilcoxon
Rank Sum test.
according
to Wilcoxon
Rank Sum test.
8 68.81 38.06 0.68 5.40
f 9.74 + 22.09 zt 0.28b f 2.03’
y-2.33-0.07x
weight of tumor / rat (g)
Fig. 1. Negative correlation of tumor weight and hepatic GSSG/(GSH+GSSG)% in the 50 kcal and 45% fat dietary group (I). The confidence interval is at the level of P = 0.01. Linear correlation coefficient is r = - 0.64.
In accordance with our results, several studies have demonstrated an inhibitory effect of calorie restriction during the late stages of carcinogenesis both in mice and rats [20,31,32,34]. Kritchevsky et al. [19] found that 25% calorie restriction inhibited growth of mammary tumors in rats treated with 7,12-dimethylbenz[a]anthracene when the restriction was maintained for the entire fourmonth study or only during the first or last 2 months. However, we did not find reports on effects of dietary restriction on the development of established mammary tumor. An enhanced survival of ascites tumor-bearing rats by short-term mild dietary restriction was reported by Siegel et al. [26]. The investigation of glutathione in livers and tumors in the present study showed that hepatic GSH tended to be lower in the calorie restricted groups, in which reduced body weight gain and slower tumor growth were observed. Fat reduction had no effect on hepatic GSH content. Hepatic GSSG % , as an indicator of oxidative stress, was significantly higher in the calorie restricted groups. It is well known that GSH has an important function in
destroying oxygen intermediates and free radicals which are constantly formed in metabolism, and in larger amounts after administration of certain drugs, oxygen and Xrays [22]. Lower levels of peroxidizability of kidney microsomes, as well as lower levels of H202 production and lipid hydroperoxide in liver microsomes can be achieved by food restriction [37]. Thus, the lower hepatic GSH in the low calorie groups found in the present study could be a response to reduced cell damage caused by reduced peroxide formation. Alternatively, this trend may also be due to reduced extrahepatic stress (tumor burden) by calorie restriction. The higher GSSG % values in the liver observed in the calorie restricted groups could be an indicator of reduced transport of GSH from liver. The negative correlation between hepatic GSSG % and tumor weight could (Fig. 1) supports this suggestions. Tumor GSH levels tended to be lower in both low fat groups, independently of calorie levels. This might represent a response to the reduced lipid peroxidation caused by reduced fat intake, since dietary fat might alter the composition of the tumor tissue [36] and lipid content of the cell membrane [9]. A positive correlation between tumor GSH level and tumor weight was observed in the group II (r = 0.86, data not shown). This tendency could not be found in other dietary groups, a discrepancy which has to be explained in a future study. Both liver and tumor GSSG levels seemed to be insensitive to dietary change (calorie and fat) studied here. The GSSG% was 2-fold higher in tumor tissue than in liver. This could imply a higher oxidative stress in tumor than in liver tissue, the lower GSH content in tumor found in our study also supports this anticipation. However, the exact mechanisms of distribution of GSH and GSSG in liver and tumor, as well as their relationship with calorie intake and tumor development remains elusive. Altogether, the present data show that even after tumor manifestation, mild calorie restriction can inhibit growth of mammary carcinoma in rats and has an apparently greater effect
151
than dietary fat reduction. The fact that inhibition of tumor growth was most effective in the low calorie and low fat group, and glutathione status in liver was related to calorie and in tumor to fat levels, indicates that calories might play a general role in host metabolism, whereas fat might have a specific effect on tumor tissue in mammary tumor development.
Potentialtion 12
13
References 1 2
3
4
5
6
7
8
9
10
11
Albanes. D. (1987) Calorie intake, body weight, and cancer: a review. Nun. Cancer, 9, 199-217. Anderson, M.E. (1985) Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol., 113. 548-552. Angres. G. and Beth M. (1990) Effects of dietary constituents on carcinogenesis in different tumor models: An overview from 1975 to 1988. ICPEMC (International Commission For Protection Against Enviromental Mutation And Carcinogens) Working Paper No. 8, in press. Beth, M., Berger. M.R., Aksoy. M. and Schmahl. D. (1987) Comparison between the effects of dietary fat level and of calorie intake on methylnitrosourea-induced mammary carcinogenesis in femal S.D. rats. fnt. J. Cancer, 39, 737-744. Butler R.N. (1977) Testimony before the Senate Select Committee on Nutrition and Human Needs. Nutrition and Aging. US, DHEW, 6. Chan, P. and Cohen, L. (1975) Dietary fat and growth promotion of rat mammary tumors. Cancer Res.. 35, 3384-3386. Clinton, S.K., Alster, J.M.. Imrey, P.B., Simon, J. and
in rats by dietary protein
or
Review. Cancer Res.. 50, 5710-5719. Griffith, O.W. (1980) Determination of glutathione glutathione disulfide using glutathione reductase 2vinylpyridine. Anal. Biochem.. 106, 207-212.
and and
14
Hendrich. S. and Pitot. H.C. (1987) Enzymes of glutathione metabolism as biochemical markers during hepatocarcinogenesis. Cancer Metastasis Rev., 6, 155-178.
15
Hollander, M. and Wolfe, D.A. (1973) Nonparametric statistical methods. John Wiley, New York. Ip, C. and Ip. M.M. (1980) Inhibition of mammary tumorigenesis by a reduction of fat intake after carcinogen
Acknowlegements
The authors thank K. Klimo, R. Bechtel, M. Konig, M. Bucur, A. Danisman and C. Bischof for their technical assistance.
of oxygentoxicity
aminoacid deficiency. J. Appl. Physiol., 54, 147- 151. Freedman, L.S.. Clifford, C. and Messina, M. (1990) Analysis of dietary fat, calorie, body weight, and the development of mammary tumors in rats and mice: A
16
17
18
19
20
21
22
Visek W. J. (1988) The combined effects of dietary protein and fat intake during the promotion phase of 7,12-dimethylbenz(a)anthracene-induced breast cancer in rats. J. Nun., 118, 1577-1583. Cohen. L.A., Choi. K.W. and Wang C.X. (1988) Influence of dietary fat, calorie restriction, and voluntary exmammary n-nitrosomethylurea-induced ercise on tumorigenesis in rats. Cancer Res.. 48, 4276-4283.
23
Cohen, L.A. (1981) Mechanisms by which dietary fat may stimulate carcinogenesis in experimental animals. Cancer Res., 41, 3808-3810. Coles, B. and Ketterer, B. (1990) The role of glutathione and glutathione transferase in chemical carcinogenesis. Crit. Rev. Biochem. Mol. Biol., 25, 47-70. Deneke, S.M.. Gershoff. S.N. and Fanburg, B.L. (1983)
26
24 25
27
treatment in young versus adult rats. Cancer Lett., 11, 35-42. Ketterer. B. (1988) Protective role of glutathione and glutathione transferases in mutagenesis and carcinogenesis. Mutat. Res.. 202, 343-361. Klurfeld. D.M., Welch, C.B., Davis, M. J. and Kritchevsky, D. (1989) Determination of degree of energy restriction reduce necessary to DMBA-induced mammary tumorigenesis in rats during the promotion phase. J. Nutr.. 119. 286-291. Kritchevsky, D.. Welch, C.B. and Klurfeld. D.M. (1989) Response of mammary tumors to caloric restriction for different time periods during the promotion phase. Nutr. Cancer, 12, 259-269. Kritchevsky, D.. Weber. M.M., Buck, C.L. and Klurfeld. D.M. (1984) Dietary fat versus caloric content in initiation and promotion of 7.12-dimethyl-benz(a)anthracene induced mammary tumorigenesis in rats. Cancer Res., 44. 3174-3177. Larsson A. Orrenius S, Holmgren A, Mannervik B (eds) Functions of glutathione: (1983) biochemical, physiological, toxicological and clinical aspects. pp. 9- 10, 40, 131-132. 256-258, 320-322. Raven Press, New York. Meister, A. and Anderson, M.E. (1983) Glutathione. Ann. Rev. Biochem.. 52, 711-760. Novi. A.M. (1981) Regression of Aflatoxin Bl-induced hepatocellular carcinomas by reduced glutathione. Science, 212, 541-542. Sachs, L. (1983) Angewante Statistik. 6. Auflage, Springer Verlag Berlin, Heidelberg-New York-Tokyo. Sate, K. (1989) Glutathione transferase as a markers of preneoplasia and neopfasia. Adv. Cancer Res.. 52, 205-255. Siegel, 1.. Liu, T.L.. Nepomuceno. N. and Gleicher, N. (1988) Effects of short-term dietary restriction on survival of mammary ascites tumor-bearing rats. Cancer Invest., 6, 677-680. Sies. H. (1989) Zur Biochemie der Thiogruppe: Bedeutung des Glutathions. Naturwissenschaft. 76, 57-64.
152
28
Simopoulo, S. (1987) Nutritional cancer risks derived from energy and fat. Med. Oncol. Tumor Pharmacother., 4. 227-239.
33
29
34
30
Sinha, D.K., Gebhard, R.L. and Pazik, J.E. (1988) Inhibition of mammary carcinogenesis in rats by dietary restriction. Cancer L&t., 40, 133-141. Sylvester, P.W.. Ip. C. and Ip. M.M. (1986) Effectsof high
35
31
dietary fat on the growth and development of ovarianindependent carcinogen-induced mammary tumors in rats. Cancer Res.. 6, 736-769. Tannenbaum, A. (1944) The dependence of the genesis of
32
induced skin tumors on the calorie intake during different stages of carcinogenesis. Cancer Res., 4, 673-677. Tannenbaum, A. (1940) The initiation and growth of tumors: Introduction: I. Effects of underfeeding. Am. J. Cancer, 38, 335-350.
36
37
Weindruch, R. and Walford. R.L. (1988) The retardation of aging and disease by dietary restriction. pp.31--100. Charles C Thomas Publisher, Springfield-Illinois-U.S.A. Weindruch. R. and Walford, R.L. (1982) Dietary restriction in mice beginning at 1 year of age: effect on lige-span and spontaneous cancer incidence. Science 215. 1415-1418. Welsch, C.W. (1987) Enhancement of mammary tumorigenesis by dietary fat: Review of potential mechanisims. Am. J. Clin. Nutr., 45, 192-202. Wicha, M.S., Liotta. L.A. and Kidwell, W.R. (1979) Effects of free fatty acids on the growth of normal and neoplastic rat mammary epithelial cells. Cancer Res., 39. 426-435. Yu, B.P., Lee, D.W., Marler, C.G. and Choi, J.H. (1990) Proc. Soci. Exp. Biol. Med., 193. 13-15.
Cancer Letters. 57 (1991) 145- 152 Elsevier Scientific Publishers Ireland Ltd.
Effect of dietary calorie and fat restriction on mammary tumor growth and hepatic as well as tumor glutathione in rats P. Zhu, E. Frei, B. Bunk, Institute of Toxicology Heidelberg
M.R. Berger and D. Schm8hla
and Chemotherapy,
German
Cancer
(Received
5 February
1991)
(Accepted
5 February
1991)
Summary Effect of dietary calorie restriction and fat reduction on growth of established mammary carcinoma in rats and on glutathione levels in liuer and tumor tissue was investigated. Reduced (GSH) and oxidized (GSSG) glutathione were determined enzymatically. Female Sprague-Dawley rats were injected with 25 mg/kg methylnitrosourea (MNU) on day 50 of life for tumor induction, and subsequently fed a diet containing 50 kcal/day with 45% (energy 48) fat. When tumors reached approximately 1 cm3, the diet was changed for 10 A 2 weeks.
Four dietary groups were formed: two high calorie groups (50 kcal/day) with 45% or 25 !%Ifat and two calorie restricted groups (35 kcal/day) with 45% or 25% fat, respectively. Tumor growth was significantly inhibted by the 30 % calorie restriction, and the inhibition
restricted reduction
was most effective in the calorie group with low fat level. However, of fat, alone, had no significant in-
hibitory effect. GSSG levels in both liver and tumor showed no differences among the groups. Hepatic GSH levels tended to be lower showed
Research
Center,
groups
with
Im Neuenheimer
Feld
280.
D-6900
(F.R.G.)
in the no
calorie-restricted difference
groups,
between
Correspondence to: M.R. Berger, Institute of Toxicology German Cancer Research Center, Chemotherapy, Neuenheimer Feld 280, D-6900 Heidelberg, F.R.G.
0304-3835/91/$03.50 Published and Printed
and
isocaloric and Im
0 1991 El sevier Scientific Publishers in Ireland
different
fat
levels.
In
contrast,
GSH in tumor tissue tended to be lower in the low fat groups, independently of caiorie levels.
Keywords: glutathione; mammary carcinoma
dietary
restriction;
Introduction The effect of different dietary calorie levels and/or fat intake on mammary tumorigenesis in rats has been intensively investigated [4,6-8,16,18,19,29,30,32,35]. Freedman et al. [12] analysed data from 100 animal experiments and reported that both high calorie and high fat intake independently increased mammary tumor incidence, and that the effect of fat was only 2/3 of the calorie effect both in SpragueDawley rats and in mice. Reviews of animal studies on the relationship between fat intake and mammary tumor incidence by other authors yielded inconsistent and inconclusive results [3,28]. The mechanisms by which dietary restriction prolongs life and inhibits age-associated diseases [33], including many cancers [l], are still not clear. Dietary restriction could be involved in the processes regulating cellular detoxification and redox systems against oxidative damage. It has been reported that diet can change the GSH level and the activity of Ireland Ltd.
146
GSH-related enzymes in liver and other tissues [ 11,2 11. Correlations between GSH and/or GSH-related enzymes and carcinogenesis have been reported in many studies [10,14,17,23,25]. GSH is co-substrate in the GSH-peroxidase reaction for the reduction of hydroperoxides in the defense against oxidative stress [27]. Hence, an alteration in the glutathione redox status could have an influence on the tumor growth, and/or, indicate metabolic disturbances contributing to tumor progression. A life-long dietary restriction as a method for prevention of cancer is not very realistic in the human situation. We therefore investigated the influence of calorie restriction and fat content, on the growth of methylnitrosourea (MNU)-induced mammary tumors after their manifestation. The status of glutathione in the liver and in the mammary tumors, and its possible correlation with tumor growth in the different calorie and fat groups were also studied. Materials and Methods Animals Female Sprague-Dawley rats were purchased from the Zentalinstitut ftir Versuchstierzucht, Hannover, F.R.G., at the age of 40 days. The whole experiment lasted 30 weeks. Chemicals
and reagents
MNU was synthesized in our institute (M. Na2HP04, Na,EDTA, Wiessler) . NaH2P04, 5-sulfosalicylic acid, 2-vinylpyridine stabilized with 0.1% 4-tert-butylbenzcatechin, triethanolamine and Tris(hydroxymethyl)-aminomethan were supplied by Merck, Darmstadt, F.R.G. 5,5’-Dithio-bis(-2-nitrobenzoic acid) (DTNB, Ellman’s reagent), GSH, GSSG, NADPH and glutathione reductase (600 (98%) units/ml) were purchased from Boehringer Mannheim GmbH, Mannheim, F.R.G. Bovine serum albumin (BSA) was supplied by Serva, Heidelberg, F.R.G. Bio-Rad Protein Assay Kit was purchased by Bio-Rad Laboratories GmbH, Munich, F.R.G.
Mammary
tumor
induction
Rats (116) were injected via the tail vein with 25 mg/kg MNU on day 50 of life to induce mammary carcinoma. Diet and feeding
Each rat was placed in an individual cage in a temperature (21 f Z’C), light (12-h light, 12-h dark) and humidity (60%) controlled room. The animals were given tap water ad libitum. Prior to tumor induction, animals were fed a standard commercial diet (Altromin 1320, Lage, F.R.G.). One day after MNU injection, all the rats were fet a diet containing 50 kcal/day and 45% (energy %a) fat. When tumors reached approximately 1 cm3, animals were randomized into the following four dietary groups for 10 f 2 weeks: group I: 50 kcal/day 45% fat (high calorie and high fat); group 11: 35 kcal/day 45% fat (calorie restricted); group III: 50 kcal/day 25% fat (high calorie and fat reduced); group IV: 35 kcal/day 25% fat (calorie restricted and fat reduced). The composition of diets is shown in Table I. All diets were obtained from Unilever Research, Rotterdam, Netherlands, kept at - 20°C and thawed before use. Rats were fed every morning and weighed and palpated for tumors weekly. Liver
and tumor
preparation
At the end of the experiment, rats were exsanguinated under ether anesthesia and a fraction of liver tissue was removed after perfusion with 0.9% NaCl. All tumors were excised, weighed and cleaned from connective and necrotic tissue. Parts of the tumors were fixed in 10% formalin for histological examination (D. Komitowski and F. Amelung, Institute of Experimental Pathology, German Cancer Heidelberg, F.R.G.). Research Center, Prepared remaining tissues were immediately frozen in liquid nitrogen and stored at - 70°C until assayed. All the samples were collected at 1200 h to minimize a possible diurnal fluctuation of glutathione.
147 Table
1.
Dietary
groups
and
composition
ND25”
ND45”
Diet kcal/day (Group
(l) 35
no.)
Composition:
g/100
of diets.
(II)
50
(III) 35
g
27.80
23.90
Corn
starch
41.00
57.40
Palm
oil
16.28
7.80
3.04
1.46
Casein
Lard
2.39
1.14
Cellulose
6.70
5.80
Vitamins
0.45
0.39
Minerals
2.42
2.08
24.00
24.00
31.00
51.00
45.00
25.00
15.00
15.00
Sunflower
Protein
(%)h
(%)
Carbohydrate Fat Fiber
oil
seed
(W) (mg/kcal)
Vitamins
(mg/kcal)
1.00
1.00
Minerals
(mg/kcal)
5.40
5.40
“ND = normal for group diets
West
German
bPercentage
diet; ND45
for group
III and IV; the fat composition
thetic
were
(IV)
according
to the
I and II. and ND25 of these
fat profile
semisyn-
of ‘normal’
diet. of total
specificity. Briefly, every test cuvette contained 700 ~1 NADPH (0.3mM) in phosphate buffer phosphate, 6.3 mM (143 mM sodium Na2EDTA, pH 7.5), 100 ~1 DTNB (6 mM) in phosphate buffer, 175 ~1 HP0 and 25 ~1 sample. Change in absorption was determined following addition of 10 ~1 glutathione reductase (266 U/ml in phosphate buffer) in triplicate using a Perkin-Elmer Lambda 5 UV/VIS Photometer at 412 nm. The reference cuvette contained 25 ~1 5% 5-sulfosalicylic acid instead of sample. To determine GSSG, GSH was derivatized with 2-vinylpyridine. Triethanolamine (1~2 (w/w) diluted with H20) was added so that the pH was between 6 and 7. After 60 min reaction, GSSG determination was carried out with DTNB-GSSG reductase recycling assay as described above. Standard samples of GSH and GSSG were treated in the same way, and the amount of GSH and GSSG was calculated from the standard curve.
energy.
Determination of GSH and GSSG Liver and tumor tissues from some rats (as indicated in Tables III and IV) were powdered frozen in nitrogen with a Micro-Dismembrator (B. Braun Melsungen AG, F.R.G.) and homogenized in 5% 5-sulfosalicylic acid to precipitate protein. The homogenates were centrifuged at 4000 rev./min for 6 min at 4°C. The supernatant was used for total glutathione (GSH + GSSG, in GSH equivalents) and GSSG determination. GSH and GSSG were determined with a DNTB-GSSG reductase recycling assay according to Griffith and Anderson [2,13] with minor modifications. One fraction of liver and l-2 tumor samples per rat were assayed. All samples were diluted to 3 different concentrations between 20- 120 PM for GSH and between 0.5-8.0 PM for GSSG with 5% sulfosalicylic acid. In this range, the assay offers high sensitivity and
Determination of protein content The 5-sulfosalicylic acid precipitates were dissolved with 0.1 M NaOH at 60°C. Protein content was determined after dilution in Tris-HC1 buffer (pH 7.4) with Bio-Rad Protein Assay Kit spetrophotometrically at 595 nm. BSA was used as standard in this assay. Statistical analysis All the data were processed in the Adam system (software for statistical analysis of biomedical data, German Cancer Research Center, Heidelberg, F.R.G.). Comparisons of parameters for statistically significant differences between groups were made by the Wilcoxon Rank Sum test [15]. The mortality within the groups was compared by Fisher’s exact test [24]. Results Influence
of calorie and fat restriction
on tumor
growth
At the time of tumor manifestation
(18 * 4
148
weeks after MNU injection) 90 rats (78% of MNU injected rats) bearing a tumor volume of 1 cm3 were randomized into the 4 dietary groups. Histology of the tumors revealed 94% adenocarcinoma and 6 % adenoma. The comparison of dietary groups I and II, and groups III and IV, at 10 f 2 weeks after dietary showed that body weight, tumor change, number and tumor weight (Table II) were significantly lower in the calorie restricted groups than in the high calorie groups (P < 0.05). The difference in the ratio of tumor to body weight was still significant (P < 0.05). This indicated that the body weight was less affected by calorie restriction than the tumor weight. The lowest tumor burden was found in group IV, in which rats received low calorie diet with low fat level. Rats in the calorie restricted groups had no loss of body weight during the experiment, but a reduced body weight gain. However, isocaloric diets with reduced fat levels had no significant effect on body weight, tumor number and weight. Mortality was related neither to calorie nor to fat levels. Glutathione status Glutathione in
her Liver weight was significantly reduced by calorie restriction, but showed no differences between the 45% and 25% fat isocaloric groups (Table III). No significant differences in hepatic protein con-
Table II. Diet kcal/day
Effect of dietary calorie content
on tumor growth,
tent per g wet liver tissue among the four dietary groups were observed, indicating that reduction of fat level had no influence on liver protein content. No significant differences were found in GSSG levels. The mean GSH levels were lower in the low calorie groups, although the difference was significant only between groups III and IV. The ratio of GSSG to total glutathione in % (GSSG W) was accordingly in the low calorie groups higher than in the high calorie groups for both fat levels. Furthermore, the GSSG% in the liver was found to be inversely related to tumor weight in group I, when linear regression was used for establishment of the correlation (Fig. 1). In the other three groups coefficient of linear regression (r) was also negative, but did not reach significance. Using all data regardless of diet, the correlation was still significant (r = - 0.44). No correlation between hepatic GSH or GSSG and tumor burden of animals was detected. Glutathione
in tumor
tissue
Table IV shows the glutathione status in mammary tumor tissue. Similar to liver, no significant differences were observed in either tumor protein or GSSG levels between the groups. The GSH levels were significantly reduced in groups III and IV compared to group II (P < 0.05). As a result, the GSSG% in group IV was higher than in group II. A
liver and body weight of rats”
ND45
ND25
(Group no.)
Number of rats Tumor number/rat Tumor weight/rat (g) Body weight (g) Tumor/body weight (W) Mortality ( W)
500)
35(H)
50(111)
35(IV)
21
23
20
2.43 zt 1.12 10.87 +z 7.02 292 zt 29 3.8 zt 2.5 3/24
1.74 l 1.10 6.07 * 7.42 277 f 20 2.2 zt 2.7 l/24
2.35 zt 1.02 7.36 zt 7.03 294 f 15 2.5 +z 2.4 o/20
19 0.95 l 0.71 3.22 zt 4.10 268 zt 23 1.3 f 1.7 3/22
The results are expressed as mean f S.D. aAll parameters except mortality were significantly (P < 0.05, Wilcoxon Rank Sum test).
different
between
group
I and II and between
group
111and IV
149
Table 111. Effect of dietary calorie level on glutathione Diet kcal/day
and protein
content
ND25
ND45 (Group no.)
35(11)
50(I) Number of rats Liver weight/rat (g) Protein mg/g liver GSSG nmol/g liver GSH pmol/g liver GSSG(%)”
16 7.68 107.5 48.71 2.82 1.68
in rat liver.
19 f 0.80 zt 14.72 f 20.00 A 0.70b ztz 0.71d
6.29 103.00 48.52 2.49 1.99
The results are expressed as mean + S.D. “GSSG/(GSH + GSSG) in 46. bcP = 0.016; low calorie groups versus high calorie groups, d.eP = 0.010; low calorie groups versus high calorie groups,
similar, but insignificant difference was also found between groups 1 and III. No correlation between GSSG or GSSG% and tumor weight could be observed. Mean GSH levels in tumor tissue were approximately l/3 (26-38%) of those found in liver, whereas GSSG levels ranged between 56 and 70% of those found in liver, and consequently the GSSG% in tumor was approximately Z-fold higher than in liver.
35(IV)
5O(II!)
+z f zt f f
18 7.58 98.44 50.03 2.89 1.74
0.99 13.91 12.3 0.67c 0.51’
according according
to Wilcoxon to Wilcoxon
18 f 1.28 + 10.24 zt 21.51 A 0.87b A 0.60d
5.73 f 0.51 101.78 f 17.27 51.92 +z 22.35 2.31 ztz 0.66 2.19 f 0.73’
Rank Sum test. Rank Sum test.
In this study we used semisynthetic diets in which fatty acid composition reflected the fatty acid profile of normal West German diet, and
found that a 30% calorie restriction could inhibit the growth of MNU-induced mammary carcinoma in rats, even after tumor manifestation. The most effective inhibition occurred in the low calorie group with low fat level. However, the reduction of fat with complementary increase of carbohydrates and no calorie restriction had no significant inhibitory effect. Reduced weight gain but no weight loss was observed in calorie restricted groups, indicating that mild calorie restriction was used in this study. The development of body weight and tumor growth during the experiment will be described in more detail elsewhere (Bunk et al. in preparation).
Table IV. Effect of dietary fat level on glutathione
in mammary
Discussion
Diet kcal/day
content
ND45
tumors. ND25
(Group no.)
Number of rats Protein mg/kg tumor GSSG nmol/g tumor GSH pmol/g tumor GSSGBc
50(I)
35(1I)
11 73.63 zt 11.11 32.58 ziz 21.16 0.91 f 0.29’ 3.58 f 2.21d
8 68.84 35.07 0.95 3.46
The results are expressed as mean f S.D “.bP = 0.04; low calorie groups versus high calorie groups, ‘GSSG/(GSH + GSSG) W. d.eP c 0.03; low calorie groups versus high calorie groups,
5@(M)
ziz 14.69 f 18.15 f 0.22 ziz 1.22d
5 67.85 28.44 0.74 4.10
35(IV)
f zt + f
7.09 7.19 0.23b 1.74’
according
to Wilcoxon
Rank Sum test.
according
to Wilcoxon
Rank Sum test.
8 68.81 38.06 0.68 5.40
f 9.74 + 22.09 zt 0.28b f 2.03’
y-2.33-0.07x
weight of tumor / rat (g)
Fig. 1. Negative correlation of tumor weight and hepatic GSSG/(GSH+GSSG)% in the 50 kcal and 45% fat dietary group (I). The confidence interval is at the level of P = 0.01. Linear correlation coefficient is r = - 0.64.
In accordance with our results, several studies have demonstrated an inhibitory effect of calorie restriction during the late stages of carcinogenesis both in mice and rats [20,31,32,34]. Kritchevsky et al. [19] found that 25% calorie restriction inhibited growth of mammary tumors in rats treated with 7,12-dimethylbenz[a]anthracene when the restriction was maintained for the entire fourmonth study or only during the first or last 2 months. However, we did not find reports on effects of dietary restriction on the development of established mammary tumor. An enhanced survival of ascites tumor-bearing rats by short-term mild dietary restriction was reported by Siegel et al. [26]. The investigation of glutathione in livers and tumors in the present study showed that hepatic GSH tended to be lower in the calorie restricted groups, in which reduced body weight gain and slower tumor growth were observed. Fat reduction had no effect on hepatic GSH content. Hepatic GSSG % , as an indicator of oxidative stress, was significantly higher in the calorie restricted groups. It is well known that GSH has an important function in
destroying oxygen intermediates and free radicals which are constantly formed in metabolism, and in larger amounts after administration of certain drugs, oxygen and Xrays [22]. Lower levels of peroxidizability of kidney microsomes, as well as lower levels of H202 production and lipid hydroperoxide in liver microsomes can be achieved by food restriction [37]. Thus, the lower hepatic GSH in the low calorie groups found in the present study could be a response to reduced cell damage caused by reduced peroxide formation. Alternatively, this trend may also be due to reduced extrahepatic stress (tumor burden) by calorie restriction. The higher GSSG % values in the liver observed in the calorie restricted groups could be an indicator of reduced transport of GSH from liver. The negative correlation between hepatic GSSG % and tumor weight could (Fig. 1) supports this suggestions. Tumor GSH levels tended to be lower in both low fat groups, independently of calorie levels. This might represent a response to the reduced lipid peroxidation caused by reduced fat intake, since dietary fat might alter the composition of the tumor tissue [36] and lipid content of the cell membrane [9]. A positive correlation between tumor GSH level and tumor weight was observed in the group II (r = 0.86, data not shown). This tendency could not be found in other dietary groups, a discrepancy which has to be explained in a future study. Both liver and tumor GSSG levels seemed to be insensitive to dietary change (calorie and fat) studied here. The GSSG% was 2-fold higher in tumor tissue than in liver. This could imply a higher oxidative stress in tumor than in liver tissue, the lower GSH content in tumor found in our study also supports this anticipation. However, the exact mechanisms of distribution of GSH and GSSG in liver and tumor, as well as their relationship with calorie intake and tumor development remains elusive. Altogether, the present data show that even after tumor manifestation, mild calorie restriction can inhibit growth of mammary carcinoma in rats and has an apparently greater effect
151
than dietary fat reduction. The fact that inhibition of tumor growth was most effective in the low calorie and low fat group, and glutathione status in liver was related to calorie and in tumor to fat levels, indicates that calories might play a general role in host metabolism, whereas fat might have a specific effect on tumor tissue in mammary tumor development.
Potentialtion 12
13
References 1 2
3
4
5
6
7
8
9
10
11
Albanes. D. (1987) Calorie intake, body weight, and cancer: a review. Nun. Cancer, 9, 199-217. Anderson, M.E. (1985) Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol., 113. 548-552. Angres. G. and Beth M. (1990) Effects of dietary constituents on carcinogenesis in different tumor models: An overview from 1975 to 1988. ICPEMC (International Commission For Protection Against Enviromental Mutation And Carcinogens) Working Paper No. 8, in press. Beth, M., Berger. M.R., Aksoy. M. and Schmahl. D. (1987) Comparison between the effects of dietary fat level and of calorie intake on methylnitrosourea-induced mammary carcinogenesis in femal S.D. rats. fnt. J. Cancer, 39, 737-744. Butler R.N. (1977) Testimony before the Senate Select Committee on Nutrition and Human Needs. Nutrition and Aging. US, DHEW, 6. Chan, P. and Cohen, L. (1975) Dietary fat and growth promotion of rat mammary tumors. Cancer Res.. 35, 3384-3386. Clinton, S.K., Alster, J.M.. Imrey, P.B., Simon, J. and
in rats by dietary protein
or
Review. Cancer Res.. 50, 5710-5719. Griffith, O.W. (1980) Determination of glutathione glutathione disulfide using glutathione reductase 2vinylpyridine. Anal. Biochem.. 106, 207-212.
and and
14
Hendrich. S. and Pitot. H.C. (1987) Enzymes of glutathione metabolism as biochemical markers during hepatocarcinogenesis. Cancer Metastasis Rev., 6, 155-178.
15
Hollander, M. and Wolfe, D.A. (1973) Nonparametric statistical methods. John Wiley, New York. Ip, C. and Ip. M.M. (1980) Inhibition of mammary tumorigenesis by a reduction of fat intake after carcinogen
Acknowlegements
The authors thank K. Klimo, R. Bechtel, M. Konig, M. Bucur, A. Danisman and C. Bischof for their technical assistance.
of oxygentoxicity
aminoacid deficiency. J. Appl. Physiol., 54, 147- 151. Freedman, L.S.. Clifford, C. and Messina, M. (1990) Analysis of dietary fat, calorie, body weight, and the development of mammary tumors in rats and mice: A
16
17
18
19
20
21
22
Visek W. J. (1988) The combined effects of dietary protein and fat intake during the promotion phase of 7,12-dimethylbenz(a)anthracene-induced breast cancer in rats. J. Nun., 118, 1577-1583. Cohen. L.A., Choi. K.W. and Wang C.X. (1988) Influence of dietary fat, calorie restriction, and voluntary exmammary n-nitrosomethylurea-induced ercise on tumorigenesis in rats. Cancer Res.. 48, 4276-4283.
23
Cohen, L.A. (1981) Mechanisms by which dietary fat may stimulate carcinogenesis in experimental animals. Cancer Res., 41, 3808-3810. Coles, B. and Ketterer, B. (1990) The role of glutathione and glutathione transferase in chemical carcinogenesis. Crit. Rev. Biochem. Mol. Biol., 25, 47-70. Deneke, S.M.. Gershoff. S.N. and Fanburg, B.L. (1983)
26
24 25
27
treatment in young versus adult rats. Cancer Lett., 11, 35-42. Ketterer. B. (1988) Protective role of glutathione and glutathione transferases in mutagenesis and carcinogenesis. Mutat. Res.. 202, 343-361. Klurfeld. D.M., Welch, C.B., Davis, M. J. and Kritchevsky, D. (1989) Determination of degree of energy restriction reduce necessary to DMBA-induced mammary tumorigenesis in rats during the promotion phase. J. Nutr.. 119. 286-291. Kritchevsky, D.. Welch, C.B. and Klurfeld. D.M. (1989) Response of mammary tumors to caloric restriction for different time periods during the promotion phase. Nutr. Cancer, 12, 259-269. Kritchevsky, D.. Weber. M.M., Buck, C.L. and Klurfeld. D.M. (1984) Dietary fat versus caloric content in initiation and promotion of 7.12-dimethyl-benz(a)anthracene induced mammary tumorigenesis in rats. Cancer Res., 44. 3174-3177. Larsson A. Orrenius S, Holmgren A, Mannervik B (eds) Functions of glutathione: (1983) biochemical, physiological, toxicological and clinical aspects. pp. 9- 10, 40, 131-132. 256-258, 320-322. Raven Press, New York. Meister, A. and Anderson, M.E. (1983) Glutathione. Ann. Rev. Biochem.. 52, 711-760. Novi. A.M. (1981) Regression of Aflatoxin Bl-induced hepatocellular carcinomas by reduced glutathione. Science, 212, 541-542. Sachs, L. (1983) Angewante Statistik. 6. Auflage, Springer Verlag Berlin, Heidelberg-New York-Tokyo. Sate, K. (1989) Glutathione transferase as a markers of preneoplasia and neopfasia. Adv. Cancer Res.. 52, 205-255. Siegel, 1.. Liu, T.L.. Nepomuceno. N. and Gleicher, N. (1988) Effects of short-term dietary restriction on survival of mammary ascites tumor-bearing rats. Cancer Invest., 6, 677-680. Sies. H. (1989) Zur Biochemie der Thiogruppe: Bedeutung des Glutathions. Naturwissenschaft. 76, 57-64.
152
28
Simopoulo, S. (1987) Nutritional cancer risks derived from energy and fat. Med. Oncol. Tumor Pharmacother., 4. 227-239.
33
29
34
30
Sinha, D.K., Gebhard, R.L. and Pazik, J.E. (1988) Inhibition of mammary carcinogenesis in rats by dietary restriction. Cancer L&t., 40, 133-141. Sylvester, P.W.. Ip. C. and Ip. M.M. (1986) Effectsof high
35
31
dietary fat on the growth and development of ovarianindependent carcinogen-induced mammary tumors in rats. Cancer Res.. 6, 736-769. Tannenbaum, A. (1944) The dependence of the genesis of
32
induced skin tumors on the calorie intake during different stages of carcinogenesis. Cancer Res., 4, 673-677. Tannenbaum, A. (1940) The initiation and growth of tumors: Introduction: I. Effects of underfeeding. Am. J. Cancer, 38, 335-350.
36
37
Weindruch, R. and Walford. R.L. (1988) The retardation of aging and disease by dietary restriction. pp.31--100. Charles C Thomas Publisher, Springfield-Illinois-U.S.A. Weindruch. R. and Walford, R.L. (1982) Dietary restriction in mice beginning at 1 year of age: effect on lige-span and spontaneous cancer incidence. Science 215. 1415-1418. Welsch, C.W. (1987) Enhancement of mammary tumorigenesis by dietary fat: Review of potential mechanisims. Am. J. Clin. Nutr., 45, 192-202. Wicha, M.S., Liotta. L.A. and Kidwell, W.R. (1979) Effects of free fatty acids on the growth of normal and neoplastic rat mammary epithelial cells. Cancer Res., 39. 426-435. Yu, B.P., Lee, D.W., Marler, C.G. and Choi, J.H. (1990) Proc. Soci. Exp. Biol. Med., 193. 13-15.
