- Email: [email protected]
Attenuation of the anticarcinogenic action of selenium by vitamin E deficiency
Cancer Letters, 25 (1985) 325-331 Elsevier Scientific Publishers Ireland Ltd.
325
ATTENUATION OF THE ANTICARCINOGENIC ACTION OF SELENIUM BY VITAMIN E DEFICIENCY CLEMENTIP
Department of Breast Surgery and Breast Cancer Research Unit, Roswell Park Memorial Institute, Buffalo, NY 14263 (U.S.A.) (Received 29 October 1984) (Revised version received 23 November 1984) (Accepted 25 November 1984)
SUMMARY
The present study showed that in the dimethylbenz[a [anthracene-induced mammary tumor model in rats, the anticarcinogenic efficacy of selenium was much attenuated when the intake of vitamin E was deficient. Selenium supplementation at 2.5 mg/kg in the diet reduced the total tumor yield by 45% and 25%, respectively, in rats with an adequate or low vitamin E intake. Measurements of selected hepatic phase I and phase II detoxifying enzymes indicated that they were not responsive to changes in dietary vitamin E or selenium levels. However, a low vitamin E intake significantly increased lipid peroxidation in the mammary fat pad regardless of selenium status, suggesting that selenium supplementation was less effective under this nutritional condition and might be associated with the higher degree of OXidantstress in the cellular environment.
INTRODUCTION
Most of the studies in the literature on vitamin E and cancer have focused on using supranutritionallevels of vitamin E in various experimental models to determine whether a prophylactic response can be produced under this condition [4,15,20,21]. Little information is available on the influence of a low or an inadequate intake on malignant growth. In a previous publication, we have reported that vitamin E deficiency increases the risk of neoplastic development in the rat mammary gland, especially in animals with a high polyunsaturated fat intake [11]. It is impossible to discuss vitamin E in this context without mentioning the growing interest on the antitumorigenic property of selenium [12,16]. On account of the functional relationship between vitamin E and selenium in protecting cells against 'oxidant 0304-3835/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. PUblished and Printed in Ireland
326
stress, we have been interested in examining the interaction of these two nutrients in cancer prevention. Recently, we have shown that excess vitamin E supplementation increases the ability of selenium to inhibit the development of mammary tumors induced by 7,12~imethylbenz[a]anthracene (DMBA), although vitamin E by itself has no effect [8]. With the objective of further delineating the protective role of natural antioxidants in tumorigenesis, the present study was designed to evaluate whether the anticarcinogenic efficacy of selenium was compromised by a deficient vitamin E intake. MATERIALS AND METHODS
Female Sprague--Dawley rats were purchased from Charles River Breeding Laboratories, Wilmington, MA. They were fed a 20% com oil, casein-based synthetic diet from weaning until termination of the experiment. The composition of this diet has been described in detail previously [19], with the exception that stripped com oil was used. Four different groups were involved in the present study; they were divided on the basis of their vitamin E and selenium intakes. These 4 groups were designated as follows: adequate vitamin E/adequate selenium, adequate vitamin E/high selenium, deficient vitamin E/adequate selenium, and deficient vitamin E/high selenium. Diets that were adequate in vitamin E and selenium contained 50 mg/kg of the former and 0.1 rug/kg of the latter (NRC recommended requirement). The deficient vitamin E diet contained 10 mg/kg of vitamin E, while the high selenium diet contained 2 .5 mg/kg of selenium (as sodium selenite). Food and water were available ad libitum . Mammary tumors were induced by intragastric administration of 5 mg of DMBA at 50 days of age. There were 25 rats for each of the 4 dietary groups. The procedures of DMBA administration, tumor palpation and autopsy examination were described in detail previously [9]. Animals were killed 20 weeks after DMBA treatment. In this experiment, the tumors were not examined histologically to distinguish the adenocarcinomas from the fibroadenomas. From our previous experience, over 95 % of the tumors developed are adenocarcinomas. Another set of rats (8 per group) were used for the in vitro lipid peroxidation and enzyme assays. They were fed from weaning 1 of the 4 different diets and were killed 1 month later without DMBA treatment. Lipid peroxidation in 10% mammary tissue homogenates was measured by the thiobarbituric acid method according to the procedure described by Ohkawa et al. [18]. The principal thio barbituric acid reactant is considered to be malondialdehyde (MDA), which is produced by lipid oxidation and released upon heating the samples in an acid medium . The intensity of the red pigment formed is a reliable method of assessing the degree of lipid peroxidation in vitro [1]. Results were expressed as nmol MDA formed/g tissue, calculated from the absorbance at 532 nm using 1,1,3,3·tetramethoxypropane as the standard. The latter compound liberates MDA under the acidic conditions used in the assay.
327
Five different enzyme activities involved in both phase I and phase II detoxifications were determined in the liver. The liver homogenate was separated into the cytosol and microsome fractions as described previously [13]. The cytosol fraction was used to measure the activity of glutathione transferase, using Lchloro-Sd-dinitrobenzene as the substrate according to the method of Habig et al. [6]. The microsome preparation was Used to measure the activities of the 4 remaining enzymes. Aniline hydroxylase and aminopyrene N-demethylase were determined according to the procedures described by King and McCay [14] . Cytochrome c reductase and UDP: glucuronyl transferase were determined by the methods detailed by Gigon et al. [5] and Mills and Smith [17], respectively. RESULTS
Table 1 summarizes the results of the tumor data showing the effect of a deficient vitamin E intake on the anticarcinogenic efficacy of selenium. In rats receiving an adequate level of vitamin E in the diet (50 mg/kg}, selenium supplementation reduced by nearly one half both the tumor incidence as well as the total tumor yield (Group 1 vs. Group 2). A deficient Vitamin E intake alone resulted in a slight enhancement in mammary tumorigenesis, such that in rats with an adequate intake of selenium, the total nUmber of tumors produced increased from 81 to 109 (Group 1 vs, Group 3). However, the effectiveness of selenium supplementation in inhibiting neoplastic development was diminished in the presence of a deficient vitamin E intake. There was only a 25% reduction in the total tumor yield of the selenium supplemented group when compared to its corresponding control (l09 tumors in Group 3 vs. 83 tumors in Group 4). It can be seen from Table 1 that a deficient vitamin E intake did not significantly affect the growth of the rats. We have also examined the activities of 5 hepatic enzymes that are inVolved in xenobiotic metabolism. Three of these enzymes, aniline hydroxylase, aminopyrine N-demethylase and cytochrome c reductase, are classified TABLE 1
-
EFFECT OF A DEFICIENT VITAMIN E INTAKE ON THE ANTICARCINOGENIC EFFICACY OF SELENIUM
G roup
1 2 3 4
'M:ean ± S.D.
Dietary vitamin E (mg/kg)
Dietary selenium (mg/kg)
Tumor incidence
50 50 10 10
0.1 2.5 0 .1 2.5
19/25 10 /25 21/25 17/25
Total no . of tumors
(%)
(76) (40) (84) (68)
81 45 109 83
Final body wt
(g)&
325 ± 29 319±25 315 ± 27 314 ± 31
aValues are expressed as mean ± S.D. b nm ol p-aminophenol formed/min per mg protein. "nmol formaldehyde formed/min per mg protein. d nm ol cytochrome c reduced/min per mg protein. enmol conjugate formed/min per mg protein. f nmol p-nitrophenol conjugated/min per mg protein.
0.15 0.14 0.13 0.14
0.57 0.54 0.52 0.50
0.1 2.5 0.1 2.5
50 50 10 10
± ± ± ±
Aniline hydroxylase''
Dietary selenium (mg/kg)
Dietary vitamin E (mg/kg) 0.98 0.92 0.87 0.93
±0.30 ± 0.26 ± 0.23 ± 0.25
Aminopyrine N-demethylase c 54 57 49 52
± 11 ±12 ± 9 ± 9
Cytochrome c reductase d
712 766 670 703
± 79 ±84 ± 72 ±67
GSH transferase"
13 10 11 14
± ± ± ±
2.2 2.1 1.8 2.3
UDP glucuronyl transferase f
EFFECT OF DIETARY VITAMIN E AND SELENIUM INTAKE ON THE ACTIVITIES OF DETOXIFYING ENZYMES IN THE LIVER a
TABLE 2
t.:l 00
c.>
329 700 4) ~
:I
600
:;: 500 01
<,
"Eu .e
400
~
« 0 ~
'0
300 200
E e 100 ViI E (ppm) 50 S. (ppm) 0.1
50 2.5
10 0.1
10 2.5
Fig. 1. Effect of vitamin E and selenium intake on lipid peroxidation in the mammary fat pad.
in the phase I detoxification reactions; while glutathione transferase and DDP: glucuronyl transferase are the phase II conjugating enzymes. Animals in this experiment were fed the different vitamin E/selenium diets from Weaning for 1 month before they were killed. Results are summarized in Table 2. It can be seen that none of these enzymes showed any significant changes in activities in response to different dietary vitamin E and selenium intakes. In view of the antioxidant property of vitamin E, we proceeded to determine if a deficient vitamin E intake might affect the extent of lipid peroxidation in the mammary fat pad measured in vitro. The mammary tissues Were obtained from the same set of animals as above. As shown in Fig. 1, a deficient vitamin E intake significantly increased MDA formation regardless of selenium intake, suggesting that the degree of oxidant stress was amplified under a condition of vitamin E insufficiency. DISCUSSION
Results of this study showed that the anticarcinogenic efficacy of selenium was much attenuated when the intake of vitamin E was deficient. Furthermore, the reduced potency of selenium under this nutritional condition was associated with increased levels of tissue peroxidation. The present Observation on the interaction between vitamin E and selenium was conSistent with our earlier finding that excess vitamin E supplementation,
330
which resulted in a systemic suppression of lipid peroxidation, was able to potentiate the prophylactic action of selenium against mammary tumorigenesis [8] . The underlying mechanism behind the interaction of these two nutrients is not clear, but it appears that the effectiveness of selenium can be modulated depending on the extent of oxidant stress in the cellular environment. According to the above hypothesis, the primary role of vitamin E is to restrict the formation of peroxides by neutralizing free radicals, thus providing a more favorable climate for selenium to exert its actions. A proportionate increase in lipid peroxidation in the liver has been reported with a step-wise reduction in vitamin E intake [3]. Pursuing the above argument from a different angle, it is interesting to note that we have found previously that selenium failed to counteract completely the tumor enhancing effect of a diet rich in polyunsaturated fat, the consumption of which presumably would result in a higher peroxidative potential in the cells [10]. Rats on the high fat diet developed more tumors than those on the low fat diet at comparable levels of selenium supplementation. It is possible that a low vitamin E diet may affect DMBA metabolism, although results of the present study showed that there was little perturbation of the liver detoxifying enzymes under this nutritional condition. While a more exhaustive investigation might be desirable, suffice it to say that the enzymes that were selected and evaluated in this study are generally accepted as indicators of phase I and phase II metabolizing enzymes. Chen et al. [2] have reported that in rats fed a tocopherol-free diet, the activities of ethylmorphine N-demethylase and UDP: glucuronyl transferase were significantly reduced, but there was no effect on cytochrome c reductase and benz[a]pyrene hydroxylase. The difference between our study and the above study is that our diet contained 10 mg/kg of vitamin E, thereby ameliorating the severity of the vitamin E deficiency. Obviously, definitive evidence on the effect of vitamin E on DMBA metabolism can only be confirmed by DNA binding and adduct studies. In conclusion, it cannot be overemphasized tbftt interactions between different nutrients may very often dominate the activities of individual nutrients in modifying cancer risk. Thus not only is it important to assess the chemopreventive efficacy of single nutrients in a control setting, valuable information can be derived by examining how the individual effects can be enhanced or suppressed as a result of synergistic or antagonistic actions between nutrients. ACKNOWLEDGEMENT
The study was supported by grant CA 27706 from the National Cancer Institute, NIH. The author is grateful for the technical assistance of Cassandra Hayes.
331 REFERENCES 1 Bus, J.S. and Gibson, J.E. (1979) Lipid peroxidation and its role in toxicology. Rev. Biochem. Toxicol., 1, 125-149. 2 Chen, J., Goetchius, M.P., Campbell, T.C. and Combs, Jr., G.F. (1982) Effects of dietary selenium and vitamin E on hepatic mixed-function oxidase activities and in vivo covalent binding of aflatoxin B, in rats. J. Nutr., 112, 324-331. 3 Chen, L.H. (1973) Effect of vitamin E and selenium on tissue antioxidant status of rats. J. Nutr., 103, 503-508. 4 Cook, M.G. and McNamara, P. (1980) Effect of dietary vitamin E on dimethylhydrazine-induced colonic tumors in mice. Cancer Res., 40, 1329-1331. 5 Gigon, P.L., Gram, T.E. and Gillette, J.R. (1969) Studies on the rate of reduction of hepatic microsomal cytochrome P·450 by reduced nicotinamide adenine dinucleotide phosphate: effect of drug substrates. Mol. Pharmacol., 5, 109-112. 6 Habig, W.H., Pabst, M.J. and Jakoby, W.B. (1974) Glutathione-S-transferase. The first enzymatic step in mercapturic acid formation. J. Biol. Chem., 249, 71307139. 7 Hoekstra, W.G. (1975) Biochemical function of selenium and its relation to vitamin E. Fed. Proc., 34, 2083-2089. 8 Horvath, P.M. and Ip, C. (1983) Synergistic effect of vitamin E and selenium in the chemoprevention of mammary carcinogenesis in rats. Cancer Res., 43, 5335-5341. 9 Ip, C. (1980) Ability of dietary fat to overcome the resistance of mature female rats to 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis. Cancer Res., 40,2785-2789. 10 Ip, C. (1981) Factors influencing the anticarcinogenic efficacy of selenium in dimethylbenz(a)anthracene-induced mammary tumorigenesis in rats. Cancer Res., 41,2683-2686. 11 Ip, C. (1982) Dietary vitamin E intake and mammary carcinogenesis in rats. Carcinogenesis, 3, 1453-1456. 12 Ip, C. (1983) Selenium-mediated inhibition of mammary carcinogenesis. BioI. Trace Element Res., 5, 317-330. 13 Ip, C. (1984) Comparative effects of antioxidants on enzymes involved in glutathione metabolism. Life Sci., 34, 2501-2506. 14 King, M.M. and McCay, P.B. (1981) Studies on liver microsomes of female rats fed purified diets varying in fat content and with and without propyl gallate. Food Cosmet. Toxicol., 19, 13-17. 15 Kurek, M.P. and Gorwin, L.M. (1982) Vitamin E protection against tumor formation by transplanted murine sarcoma cells. Nutr. Cancer, 4, 128-139. 16 Medina, D. and Lane, H.W. (1983) Stage specificity of selenium-mediated inhibition of mouse mammary tumorigenesis. BioI. Trace Element Res., 5, 297-306. 17 Mills, G.T. and Smith, E.E.B. (1963) Uridine diphosphogalactose, uridine triphosphate, and uridine diphosphoglucuronic acid. In: Methods of Enzymatic Analysis, pp. 581-595. Editor: H.U. Bergmeyer. Academic Press, New York. 18 Ohkawa, H., Ohishi, N. and Yagi, K. (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem., 95, 351-358. 19 Selenskas, S.L., Ip, M.M. and Ip, C. (1984) Similarity between trans fat and saturated fat in the modification of rat mammary carcinogenesis. Cancer Res., 44,1321-1326. 20 Shklar, G. (1982) Oral mucosal carcinogenesis in hamsters: inhibition by vitamin E. J. Natl. Cancer Inst., 68, 791-797. 21 Slaga, T.J. and Bracken, W.M. (1977) The effects of antioxidants on skin tumor initiation and aryl hydrocarbon hydroxylase. Cancer Res., 37,1631-1635.
325
ATTENUATION OF THE ANTICARCINOGENIC ACTION OF SELENIUM BY VITAMIN E DEFICIENCY CLEMENTIP
Department of Breast Surgery and Breast Cancer Research Unit, Roswell Park Memorial Institute, Buffalo, NY 14263 (U.S.A.) (Received 29 October 1984) (Revised version received 23 November 1984) (Accepted 25 November 1984)
SUMMARY
The present study showed that in the dimethylbenz[a [anthracene-induced mammary tumor model in rats, the anticarcinogenic efficacy of selenium was much attenuated when the intake of vitamin E was deficient. Selenium supplementation at 2.5 mg/kg in the diet reduced the total tumor yield by 45% and 25%, respectively, in rats with an adequate or low vitamin E intake. Measurements of selected hepatic phase I and phase II detoxifying enzymes indicated that they were not responsive to changes in dietary vitamin E or selenium levels. However, a low vitamin E intake significantly increased lipid peroxidation in the mammary fat pad regardless of selenium status, suggesting that selenium supplementation was less effective under this nutritional condition and might be associated with the higher degree of OXidantstress in the cellular environment.
INTRODUCTION
Most of the studies in the literature on vitamin E and cancer have focused on using supranutritionallevels of vitamin E in various experimental models to determine whether a prophylactic response can be produced under this condition [4,15,20,21]. Little information is available on the influence of a low or an inadequate intake on malignant growth. In a previous publication, we have reported that vitamin E deficiency increases the risk of neoplastic development in the rat mammary gland, especially in animals with a high polyunsaturated fat intake [11]. It is impossible to discuss vitamin E in this context without mentioning the growing interest on the antitumorigenic property of selenium [12,16]. On account of the functional relationship between vitamin E and selenium in protecting cells against 'oxidant 0304-3835/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. PUblished and Printed in Ireland
326
stress, we have been interested in examining the interaction of these two nutrients in cancer prevention. Recently, we have shown that excess vitamin E supplementation increases the ability of selenium to inhibit the development of mammary tumors induced by 7,12~imethylbenz[a]anthracene (DMBA), although vitamin E by itself has no effect [8]. With the objective of further delineating the protective role of natural antioxidants in tumorigenesis, the present study was designed to evaluate whether the anticarcinogenic efficacy of selenium was compromised by a deficient vitamin E intake. MATERIALS AND METHODS
Female Sprague--Dawley rats were purchased from Charles River Breeding Laboratories, Wilmington, MA. They were fed a 20% com oil, casein-based synthetic diet from weaning until termination of the experiment. The composition of this diet has been described in detail previously [19], with the exception that stripped com oil was used. Four different groups were involved in the present study; they were divided on the basis of their vitamin E and selenium intakes. These 4 groups were designated as follows: adequate vitamin E/adequate selenium, adequate vitamin E/high selenium, deficient vitamin E/adequate selenium, and deficient vitamin E/high selenium. Diets that were adequate in vitamin E and selenium contained 50 mg/kg of the former and 0.1 rug/kg of the latter (NRC recommended requirement). The deficient vitamin E diet contained 10 mg/kg of vitamin E, while the high selenium diet contained 2 .5 mg/kg of selenium (as sodium selenite). Food and water were available ad libitum . Mammary tumors were induced by intragastric administration of 5 mg of DMBA at 50 days of age. There were 25 rats for each of the 4 dietary groups. The procedures of DMBA administration, tumor palpation and autopsy examination were described in detail previously [9]. Animals were killed 20 weeks after DMBA treatment. In this experiment, the tumors were not examined histologically to distinguish the adenocarcinomas from the fibroadenomas. From our previous experience, over 95 % of the tumors developed are adenocarcinomas. Another set of rats (8 per group) were used for the in vitro lipid peroxidation and enzyme assays. They were fed from weaning 1 of the 4 different diets and were killed 1 month later without DMBA treatment. Lipid peroxidation in 10% mammary tissue homogenates was measured by the thiobarbituric acid method according to the procedure described by Ohkawa et al. [18]. The principal thio barbituric acid reactant is considered to be malondialdehyde (MDA), which is produced by lipid oxidation and released upon heating the samples in an acid medium . The intensity of the red pigment formed is a reliable method of assessing the degree of lipid peroxidation in vitro [1]. Results were expressed as nmol MDA formed/g tissue, calculated from the absorbance at 532 nm using 1,1,3,3·tetramethoxypropane as the standard. The latter compound liberates MDA under the acidic conditions used in the assay.
327
Five different enzyme activities involved in both phase I and phase II detoxifications were determined in the liver. The liver homogenate was separated into the cytosol and microsome fractions as described previously [13]. The cytosol fraction was used to measure the activity of glutathione transferase, using Lchloro-Sd-dinitrobenzene as the substrate according to the method of Habig et al. [6]. The microsome preparation was Used to measure the activities of the 4 remaining enzymes. Aniline hydroxylase and aminopyrene N-demethylase were determined according to the procedures described by King and McCay [14] . Cytochrome c reductase and UDP: glucuronyl transferase were determined by the methods detailed by Gigon et al. [5] and Mills and Smith [17], respectively. RESULTS
Table 1 summarizes the results of the tumor data showing the effect of a deficient vitamin E intake on the anticarcinogenic efficacy of selenium. In rats receiving an adequate level of vitamin E in the diet (50 mg/kg}, selenium supplementation reduced by nearly one half both the tumor incidence as well as the total tumor yield (Group 1 vs. Group 2). A deficient Vitamin E intake alone resulted in a slight enhancement in mammary tumorigenesis, such that in rats with an adequate intake of selenium, the total nUmber of tumors produced increased from 81 to 109 (Group 1 vs, Group 3). However, the effectiveness of selenium supplementation in inhibiting neoplastic development was diminished in the presence of a deficient vitamin E intake. There was only a 25% reduction in the total tumor yield of the selenium supplemented group when compared to its corresponding control (l09 tumors in Group 3 vs. 83 tumors in Group 4). It can be seen from Table 1 that a deficient vitamin E intake did not significantly affect the growth of the rats. We have also examined the activities of 5 hepatic enzymes that are inVolved in xenobiotic metabolism. Three of these enzymes, aniline hydroxylase, aminopyrine N-demethylase and cytochrome c reductase, are classified TABLE 1
-
EFFECT OF A DEFICIENT VITAMIN E INTAKE ON THE ANTICARCINOGENIC EFFICACY OF SELENIUM
G roup
1 2 3 4
'M:ean ± S.D.
Dietary vitamin E (mg/kg)
Dietary selenium (mg/kg)
Tumor incidence
50 50 10 10
0.1 2.5 0 .1 2.5
19/25 10 /25 21/25 17/25
Total no . of tumors
(%)
(76) (40) (84) (68)
81 45 109 83
Final body wt
(g)&
325 ± 29 319±25 315 ± 27 314 ± 31
aValues are expressed as mean ± S.D. b nm ol p-aminophenol formed/min per mg protein. "nmol formaldehyde formed/min per mg protein. d nm ol cytochrome c reduced/min per mg protein. enmol conjugate formed/min per mg protein. f nmol p-nitrophenol conjugated/min per mg protein.
0.15 0.14 0.13 0.14
0.57 0.54 0.52 0.50
0.1 2.5 0.1 2.5
50 50 10 10
± ± ± ±
Aniline hydroxylase''
Dietary selenium (mg/kg)
Dietary vitamin E (mg/kg) 0.98 0.92 0.87 0.93
±0.30 ± 0.26 ± 0.23 ± 0.25
Aminopyrine N-demethylase c 54 57 49 52
± 11 ±12 ± 9 ± 9
Cytochrome c reductase d
712 766 670 703
± 79 ±84 ± 72 ±67
GSH transferase"
13 10 11 14
± ± ± ±
2.2 2.1 1.8 2.3
UDP glucuronyl transferase f
EFFECT OF DIETARY VITAMIN E AND SELENIUM INTAKE ON THE ACTIVITIES OF DETOXIFYING ENZYMES IN THE LIVER a
TABLE 2
t.:l 00
c.>
329 700 4) ~
:I
600
:;: 500 01
<,
"Eu .e
400
~
« 0 ~
'0
300 200
E e 100 ViI E (ppm) 50 S. (ppm) 0.1
50 2.5
10 0.1
10 2.5
Fig. 1. Effect of vitamin E and selenium intake on lipid peroxidation in the mammary fat pad.
in the phase I detoxification reactions; while glutathione transferase and DDP: glucuronyl transferase are the phase II conjugating enzymes. Animals in this experiment were fed the different vitamin E/selenium diets from Weaning for 1 month before they were killed. Results are summarized in Table 2. It can be seen that none of these enzymes showed any significant changes in activities in response to different dietary vitamin E and selenium intakes. In view of the antioxidant property of vitamin E, we proceeded to determine if a deficient vitamin E intake might affect the extent of lipid peroxidation in the mammary fat pad measured in vitro. The mammary tissues Were obtained from the same set of animals as above. As shown in Fig. 1, a deficient vitamin E intake significantly increased MDA formation regardless of selenium intake, suggesting that the degree of oxidant stress was amplified under a condition of vitamin E insufficiency. DISCUSSION
Results of this study showed that the anticarcinogenic efficacy of selenium was much attenuated when the intake of vitamin E was deficient. Furthermore, the reduced potency of selenium under this nutritional condition was associated with increased levels of tissue peroxidation. The present Observation on the interaction between vitamin E and selenium was conSistent with our earlier finding that excess vitamin E supplementation,
330
which resulted in a systemic suppression of lipid peroxidation, was able to potentiate the prophylactic action of selenium against mammary tumorigenesis [8] . The underlying mechanism behind the interaction of these two nutrients is not clear, but it appears that the effectiveness of selenium can be modulated depending on the extent of oxidant stress in the cellular environment. According to the above hypothesis, the primary role of vitamin E is to restrict the formation of peroxides by neutralizing free radicals, thus providing a more favorable climate for selenium to exert its actions. A proportionate increase in lipid peroxidation in the liver has been reported with a step-wise reduction in vitamin E intake [3]. Pursuing the above argument from a different angle, it is interesting to note that we have found previously that selenium failed to counteract completely the tumor enhancing effect of a diet rich in polyunsaturated fat, the consumption of which presumably would result in a higher peroxidative potential in the cells [10]. Rats on the high fat diet developed more tumors than those on the low fat diet at comparable levels of selenium supplementation. It is possible that a low vitamin E diet may affect DMBA metabolism, although results of the present study showed that there was little perturbation of the liver detoxifying enzymes under this nutritional condition. While a more exhaustive investigation might be desirable, suffice it to say that the enzymes that were selected and evaluated in this study are generally accepted as indicators of phase I and phase II metabolizing enzymes. Chen et al. [2] have reported that in rats fed a tocopherol-free diet, the activities of ethylmorphine N-demethylase and UDP: glucuronyl transferase were significantly reduced, but there was no effect on cytochrome c reductase and benz[a]pyrene hydroxylase. The difference between our study and the above study is that our diet contained 10 mg/kg of vitamin E, thereby ameliorating the severity of the vitamin E deficiency. Obviously, definitive evidence on the effect of vitamin E on DMBA metabolism can only be confirmed by DNA binding and adduct studies. In conclusion, it cannot be overemphasized tbftt interactions between different nutrients may very often dominate the activities of individual nutrients in modifying cancer risk. Thus not only is it important to assess the chemopreventive efficacy of single nutrients in a control setting, valuable information can be derived by examining how the individual effects can be enhanced or suppressed as a result of synergistic or antagonistic actions between nutrients. ACKNOWLEDGEMENT
The study was supported by grant CA 27706 from the National Cancer Institute, NIH. The author is grateful for the technical assistance of Cassandra Hayes.
331 REFERENCES 1 Bus, J.S. and Gibson, J.E. (1979) Lipid peroxidation and its role in toxicology. Rev. Biochem. Toxicol., 1, 125-149. 2 Chen, J., Goetchius, M.P., Campbell, T.C. and Combs, Jr., G.F. (1982) Effects of dietary selenium and vitamin E on hepatic mixed-function oxidase activities and in vivo covalent binding of aflatoxin B, in rats. J. Nutr., 112, 324-331. 3 Chen, L.H. (1973) Effect of vitamin E and selenium on tissue antioxidant status of rats. J. Nutr., 103, 503-508. 4 Cook, M.G. and McNamara, P. (1980) Effect of dietary vitamin E on dimethylhydrazine-induced colonic tumors in mice. Cancer Res., 40, 1329-1331. 5 Gigon, P.L., Gram, T.E. and Gillette, J.R. (1969) Studies on the rate of reduction of hepatic microsomal cytochrome P·450 by reduced nicotinamide adenine dinucleotide phosphate: effect of drug substrates. Mol. Pharmacol., 5, 109-112. 6 Habig, W.H., Pabst, M.J. and Jakoby, W.B. (1974) Glutathione-S-transferase. The first enzymatic step in mercapturic acid formation. J. Biol. Chem., 249, 71307139. 7 Hoekstra, W.G. (1975) Biochemical function of selenium and its relation to vitamin E. Fed. Proc., 34, 2083-2089. 8 Horvath, P.M. and Ip, C. (1983) Synergistic effect of vitamin E and selenium in the chemoprevention of mammary carcinogenesis in rats. Cancer Res., 43, 5335-5341. 9 Ip, C. (1980) Ability of dietary fat to overcome the resistance of mature female rats to 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis. Cancer Res., 40,2785-2789. 10 Ip, C. (1981) Factors influencing the anticarcinogenic efficacy of selenium in dimethylbenz(a)anthracene-induced mammary tumorigenesis in rats. Cancer Res., 41,2683-2686. 11 Ip, C. (1982) Dietary vitamin E intake and mammary carcinogenesis in rats. Carcinogenesis, 3, 1453-1456. 12 Ip, C. (1983) Selenium-mediated inhibition of mammary carcinogenesis. BioI. Trace Element Res., 5, 317-330. 13 Ip, C. (1984) Comparative effects of antioxidants on enzymes involved in glutathione metabolism. Life Sci., 34, 2501-2506. 14 King, M.M. and McCay, P.B. (1981) Studies on liver microsomes of female rats fed purified diets varying in fat content and with and without propyl gallate. Food Cosmet. Toxicol., 19, 13-17. 15 Kurek, M.P. and Gorwin, L.M. (1982) Vitamin E protection against tumor formation by transplanted murine sarcoma cells. Nutr. Cancer, 4, 128-139. 16 Medina, D. and Lane, H.W. (1983) Stage specificity of selenium-mediated inhibition of mouse mammary tumorigenesis. BioI. Trace Element Res., 5, 297-306. 17 Mills, G.T. and Smith, E.E.B. (1963) Uridine diphosphogalactose, uridine triphosphate, and uridine diphosphoglucuronic acid. In: Methods of Enzymatic Analysis, pp. 581-595. Editor: H.U. Bergmeyer. Academic Press, New York. 18 Ohkawa, H., Ohishi, N. and Yagi, K. (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem., 95, 351-358. 19 Selenskas, S.L., Ip, M.M. and Ip, C. (1984) Similarity between trans fat and saturated fat in the modification of rat mammary carcinogenesis. Cancer Res., 44,1321-1326. 20 Shklar, G. (1982) Oral mucosal carcinogenesis in hamsters: inhibition by vitamin E. J. Natl. Cancer Inst., 68, 791-797. 21 Slaga, T.J. and Bracken, W.M. (1977) The effects of antioxidants on skin tumor initiation and aryl hydrocarbon hydroxylase. Cancer Res., 37,1631-1635.