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Nutrition and Cancer Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/hnuc20
Ethanol calories do not enhance breast cancer in isocalorically fed C3H/Ou mice a
b
John F. Hackney , Robert W. Engelman & Robert A. Good
b
a
Department of Pharmacology, College of Medicine , University of South Florida , Tampa, FL, 33612 b
Department of Pediatrics, College of Medicine , All Children's Hospital, University of South Florida , 801 Sixth St. South, St. Petersburg, FL, 33701 Published online: 04 Aug 2009.
To cite this article: John F. Hackney , Robert W. Engelman & Robert A. Good (1992) Ethanol calories do not enhance breast cancer in isocalorically fed C3H/Ou mice, Nutrition and Cancer, 18:3, 245-253, DOI: 10.1080/01635589209514225 To link to this article: http://dx.doi.org/10.1080/01635589209514225
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/ terms-and-conditions
Ethanol Calories Do Not Enhance Breast Cancer in Isocalorically Fed C3H/Ou Mice John F. Hackney, Robert W. Engelman, and Robert A. Good
Downloaded by [] at 18:02 30 December 2014
Abstract Mammary tumorigenesis is augmented when C3H/Ou mice are fed diet ad libitum but delayed when calories are restricted by 40%. Three feeding experiments were done to evaluate the effect of ethanol on mammary tumorigenesis in isocalorically fed C3H/0u mice: 1) ad libitum feeding of semipurified solid diet, with one group receiving 12% ethanol (15 g/kg/day) in the drinking water while controls received water alone; 2) isocaloric pair feeding of semipurified solid diet, with ethanol (4 g/kg/day) administered by gavage five times per week; and 3) isocaloric pair feeding of Lieber-DeCarli liquid diet, with one group receiving 29% of calories as ethanol (20 g/kg/day) in the diet. Despite administration of ethanol to isocalorically fed C3H/Ou mice for 65 weeks by three different methods, mammary tumor development was not enhanced. In two of the three ethanol-consuming groups, weight gain and mean body weight were less in the ethanol-consuming mice than in the controls, despite equal total calorie consumption. In only one ethanol-consuming group, where mice received ethanol as a 12% solution in the drinking water, was any difference noted in the tendency to develop mammary tumors. In this case, delay in tumorigenesis was apparent in the ethanol-consuming animals (p = 0.03). These findings do not support the hypothesis that ethanol calories augment the risk for breast tumorigenesis among breast cancer-prone mice consuming isocaloric diets. Instead, reductions in weight gain and body weight among ethanol-consuming mice and an apparent reduction in mammary tumorigenesis in one of three experimental groups suggest that ethanol may decrease metabolic utilization of calories and hence contribute to lowered energy availability. This in turn could decrease tumorigenesis. (Nutr Cancer 18, 245-253, 1992)
Introduction
Numerous epidemiological studies have been performed to assess an apparent enhanced risk of developing breast cancer as a result of moderate ethanol consumption. The published results have been conflicting and controversial, but the balance of evidence appears to favor the hypothesis that ethanol increases the relative risk for breast cancer (1-8; for review, see Ref. 9). Variables that may influence breast cancer risk include diet, hormonal disposition, genetic background, possibility of viral involvement, smoking, age at onset of alcohol consumption, and frequency and amount of alcohol consumed. Because ethanol provides calories and caloric J. F. Hackney is affiliated with the Department of Pharmacology, College of Medicine, University of South Florida, Tampa, FL 33612. R. W. Engelman and R. A. Good are affiliated with the Department of Pediatrics, College of Medicine, All Children's Hospital, University of South Florida, St. Petersburg, FL 33701.
Copyright © 1992, Lawrence Erlbaum Associates, Inc.
Downloaded by [] at 18:02 30 December 2014
levels have been demonstrated to influence cancer development at multiple sites, the contribution of alcohol to breast tumorigenesis may involve its energy contribution or its influence on efficiency of utilization of total dietary calories. No succinct mechanism has been proposed to explain an etiologic relationship between alcohol and breast cancer. Consequently, animal models for breast cancer are useful for establishing whether an etiologic basis exists for the carcinogenicity of ethanol. They are also useful for determining the effect of ethanol on the nutritional status of the animal, and they provide systems in which possible mechanisms of carcinogenesis can be explored. Schrauzer and co-workers (10,11) used C3H/St mice, which develop mammary tumors spontaneously, to investigate the potential etiologic role of ethanol in breast cancer. Mice were fed a commercial diet ad libitum, with the experimental group receiving 12% ethanol in their drinking water or receiving beer in place of water and the control group receiving water alone. The time to tumorigenesis was shortened in those animals consuming 12% ethanol, but the feeding of beer in place of drinking water had no significant influence on C3H/St mammary tumor development (10,11). To more precisely quantify the relationship between alcohol consumption and the time to tumor development in the related mouse strain C3H/Ou, which develops breast cancer spontaneously, we chose to use defined semipurified diets in place of commercial chow and to ensure that both control and ethanol-consuming mice consume equal total calories. We report here that an increased rate of tumor development with ethanol consumption was not observed in isocalorically fed mice and that, under certain circumstances, ethanol may alter calorie utilization such that the rate of tumorigenesis in these mice is decreased. Materials and Methods
Animals Female C3H/Ou mice, six weeks old, were obtained from Jackson Laboratories (Bar Harbor, ME). They were housed in plastic cages and fed Purina Laboratory Chow ad libitum for one week before the beginning of the test diets. Room temperature was maintained at 24°C for the duration of the experiments. Mice were housed singly during the experiments, with the exception of the experiment in which ethanol was administered in the drinking water, where they were housed two per cage. Semipurified Diets The caloric content of the solid defined test diet was described previously (12) and provided 3.98 kcal/g, which consisted of 30.2% calories in the form of protein, 63.6% carbohydrate, and 4.5% fat (Table 1). The liquid diet, described by Lieber and DeCarli (13), was obtained from Dyets (Bethlehem, PA) and provided 1.0 kcal/ml. The caloric composition of the control form of this diet was 18% protein, 47% carbohydrate, and 35% fat. The ethanol form of the diet contained 29% of calories as ethanol (95% ethanol USP; Aaper Alcohol and Chemical, Shelbyville, KY), and the carbohydrate component was reduced to 18%. It was necessary to institute this diet gradually over a period of three weeks to overcome the inherent aversion of mice for ethanol. The ethanol diet was started with 2.5 g/100 ml ethanol; this concentration was increased by 0.5 g/100 ml each time that consumption stabilized, until the final concentration of 4 g/100 ml, which represented 29% of calories as ethanol, was achieved. With each change in ethanol concentration, the carbohydrate component was adjusted such that the sum of the calories contributed by ethanol and carbohydrate remained 47% of the total.
246
Nutrition and Cancer 1992
Table 1. Composition of Semipurifed Solid Diet Constituent Sucrose Glycerol Casein Methionine Safflower oil AIN vitamin mix AIN mineral mix Inositol Choline bitartrate Total
Downloaded by [] at 18:02 30 December 2014
Energy, kcal/g Protein/total kcal Carbohydrate/total kcal Fat/total kcal
g
kcal
47.25 16.0 29.4
189.0 64.0 117.6
2.4
0.6 2.0 1.0 3.5
18.0 3.95 1.65
0.2
0.2 0.8
100.0
397.6
0.05
3.98 0.302 0.636 0.045
Gavage Ethanol (95% USP) was mixed with Sustacal (Mead Johnson Nutritionals, Evansville, IN) to a concentration of 20% and administered (4 g/kg body wt) once per day in the morning, five days per week, Monday through Friday. Control animals were also gavaged by the same schedule with Sustacal supplemented with isocaloric sucrose in place of the ethanol. The volume gavaged did not exceed 0.7 ml/day even when the mice weighed >35 g, because greater volumes resulted in weight loss. Thus mice weighing >35 Ig received
2
60-
o E
40-
u c
3
20 -
60
20
70
Age (Weeks) Figure 1. Tendency to develop mammary tumors among isocalorically fed C3H7Ou mice in Expt 1, in which ethanol-treated mice (n = 15) consumed 12% ethanol in their drinking water and control mice (n = 15) consumed water alone. Mice of both groups consumed a semipurified solid diet described in Table 1.
248
Nutrition and Cancer 1992
40 -a—
Weight control
-•
Weight Ethanol
5 30-
Downloaded by [] at 18:02 30 December 2014
5
20
20
10
Age
30
40
(weeks)
Figure 2. Weight gain of mice fed semipunfied solid diet in Expt 1, in which mice received 12% ethanol in water or water alone. Values are means ± SEM.
(Figure 6). Mice consumed approximately 20 g/kg/day of ethanol. Weight gain in ethanol-fed animals was the same as in controls for 14 weeks and then decreased in the ethanol group in relation to the controls, despite continued pair feeding of the isocaloric diet (Figure 7). Discussion
Studies evaluating the influence of ethanol on experimental mammary tumorigenesis have been conflicting (10,11,14,15). In the experiments described here, ethanol did not enhance the rate of mammary tumor development in isocalorically fed mice despite a variety of methods of delivering ethanol to mice. In all the studies reported here, we used well-defined semipunfied solid or liquid diets and ensured isocaloric consumption. Whether mice consuming ethanol received it in the drinking water or in the semipurified liquid diet or were gavaged with ethanol and received the balance of their calories from solid diet, no substantial difference in the tendency to develop mammary tumors was noted that could be attributed to ethanol consumption. The lower weight gain with respect to energy intake and the statistically suggestive (p = 0.03) delay in mammary tumor development in ethanol-consuming mice in Experiment 1 may be attributable to reduced calorie utilization by these mice, representing a reduction in energy availability for growth and maintenance (16). This would suggest that ethanol interference with carbohydrate utilization in our first experiment may be responsible for the slight delay in the rate of tumor development in ethanol-fed animals, because they did not gain weight at the same rate as control animals despite the fact that the total calories consumed, including those contributed by ethanol, were approximately the same in the ethanol group as in the control group. If ethanol calories are excluded, then control animals consumed more solid diet calories than the ethanol group. Thus it is possible that ethanol in this study was acting
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Nutrition and Cancer Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/hnuc20
Ethanol calories do not enhance breast cancer in isocalorically fed C3H/Ou mice a
b
John F. Hackney , Robert W. Engelman & Robert A. Good
b
a
Department of Pharmacology, College of Medicine , University of South Florida , Tampa, FL, 33612 b
Department of Pediatrics, College of Medicine , All Children's Hospital, University of South Florida , 801 Sixth St. South, St. Petersburg, FL, 33701 Published online: 04 Aug 2009.
To cite this article: John F. Hackney , Robert W. Engelman & Robert A. Good (1992) Ethanol calories do not enhance breast cancer in isocalorically fed C3H/Ou mice, Nutrition and Cancer, 18:3, 245-253, DOI: 10.1080/01635589209514225 To link to this article: http://dx.doi.org/10.1080/01635589209514225
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/ terms-and-conditions
Ethanol Calories Do Not Enhance Breast Cancer in Isocalorically Fed C3H/Ou Mice John F. Hackney, Robert W. Engelman, and Robert A. Good
Downloaded by [] at 18:02 30 December 2014
Abstract Mammary tumorigenesis is augmented when C3H/Ou mice are fed diet ad libitum but delayed when calories are restricted by 40%. Three feeding experiments were done to evaluate the effect of ethanol on mammary tumorigenesis in isocalorically fed C3H/0u mice: 1) ad libitum feeding of semipurified solid diet, with one group receiving 12% ethanol (15 g/kg/day) in the drinking water while controls received water alone; 2) isocaloric pair feeding of semipurified solid diet, with ethanol (4 g/kg/day) administered by gavage five times per week; and 3) isocaloric pair feeding of Lieber-DeCarli liquid diet, with one group receiving 29% of calories as ethanol (20 g/kg/day) in the diet. Despite administration of ethanol to isocalorically fed C3H/Ou mice for 65 weeks by three different methods, mammary tumor development was not enhanced. In two of the three ethanol-consuming groups, weight gain and mean body weight were less in the ethanol-consuming mice than in the controls, despite equal total calorie consumption. In only one ethanol-consuming group, where mice received ethanol as a 12% solution in the drinking water, was any difference noted in the tendency to develop mammary tumors. In this case, delay in tumorigenesis was apparent in the ethanol-consuming animals (p = 0.03). These findings do not support the hypothesis that ethanol calories augment the risk for breast tumorigenesis among breast cancer-prone mice consuming isocaloric diets. Instead, reductions in weight gain and body weight among ethanol-consuming mice and an apparent reduction in mammary tumorigenesis in one of three experimental groups suggest that ethanol may decrease metabolic utilization of calories and hence contribute to lowered energy availability. This in turn could decrease tumorigenesis. (Nutr Cancer 18, 245-253, 1992)
Introduction
Numerous epidemiological studies have been performed to assess an apparent enhanced risk of developing breast cancer as a result of moderate ethanol consumption. The published results have been conflicting and controversial, but the balance of evidence appears to favor the hypothesis that ethanol increases the relative risk for breast cancer (1-8; for review, see Ref. 9). Variables that may influence breast cancer risk include diet, hormonal disposition, genetic background, possibility of viral involvement, smoking, age at onset of alcohol consumption, and frequency and amount of alcohol consumed. Because ethanol provides calories and caloric J. F. Hackney is affiliated with the Department of Pharmacology, College of Medicine, University of South Florida, Tampa, FL 33612. R. W. Engelman and R. A. Good are affiliated with the Department of Pediatrics, College of Medicine, All Children's Hospital, University of South Florida, St. Petersburg, FL 33701.
Copyright © 1992, Lawrence Erlbaum Associates, Inc.
Downloaded by [] at 18:02 30 December 2014
levels have been demonstrated to influence cancer development at multiple sites, the contribution of alcohol to breast tumorigenesis may involve its energy contribution or its influence on efficiency of utilization of total dietary calories. No succinct mechanism has been proposed to explain an etiologic relationship between alcohol and breast cancer. Consequently, animal models for breast cancer are useful for establishing whether an etiologic basis exists for the carcinogenicity of ethanol. They are also useful for determining the effect of ethanol on the nutritional status of the animal, and they provide systems in which possible mechanisms of carcinogenesis can be explored. Schrauzer and co-workers (10,11) used C3H/St mice, which develop mammary tumors spontaneously, to investigate the potential etiologic role of ethanol in breast cancer. Mice were fed a commercial diet ad libitum, with the experimental group receiving 12% ethanol in their drinking water or receiving beer in place of water and the control group receiving water alone. The time to tumorigenesis was shortened in those animals consuming 12% ethanol, but the feeding of beer in place of drinking water had no significant influence on C3H/St mammary tumor development (10,11). To more precisely quantify the relationship between alcohol consumption and the time to tumor development in the related mouse strain C3H/Ou, which develops breast cancer spontaneously, we chose to use defined semipurified diets in place of commercial chow and to ensure that both control and ethanol-consuming mice consume equal total calories. We report here that an increased rate of tumor development with ethanol consumption was not observed in isocalorically fed mice and that, under certain circumstances, ethanol may alter calorie utilization such that the rate of tumorigenesis in these mice is decreased. Materials and Methods
Animals Female C3H/Ou mice, six weeks old, were obtained from Jackson Laboratories (Bar Harbor, ME). They were housed in plastic cages and fed Purina Laboratory Chow ad libitum for one week before the beginning of the test diets. Room temperature was maintained at 24°C for the duration of the experiments. Mice were housed singly during the experiments, with the exception of the experiment in which ethanol was administered in the drinking water, where they were housed two per cage. Semipurified Diets The caloric content of the solid defined test diet was described previously (12) and provided 3.98 kcal/g, which consisted of 30.2% calories in the form of protein, 63.6% carbohydrate, and 4.5% fat (Table 1). The liquid diet, described by Lieber and DeCarli (13), was obtained from Dyets (Bethlehem, PA) and provided 1.0 kcal/ml. The caloric composition of the control form of this diet was 18% protein, 47% carbohydrate, and 35% fat. The ethanol form of the diet contained 29% of calories as ethanol (95% ethanol USP; Aaper Alcohol and Chemical, Shelbyville, KY), and the carbohydrate component was reduced to 18%. It was necessary to institute this diet gradually over a period of three weeks to overcome the inherent aversion of mice for ethanol. The ethanol diet was started with 2.5 g/100 ml ethanol; this concentration was increased by 0.5 g/100 ml each time that consumption stabilized, until the final concentration of 4 g/100 ml, which represented 29% of calories as ethanol, was achieved. With each change in ethanol concentration, the carbohydrate component was adjusted such that the sum of the calories contributed by ethanol and carbohydrate remained 47% of the total.
246
Nutrition and Cancer 1992
Table 1. Composition of Semipurifed Solid Diet Constituent Sucrose Glycerol Casein Methionine Safflower oil AIN vitamin mix AIN mineral mix Inositol Choline bitartrate Total
Downloaded by [] at 18:02 30 December 2014
Energy, kcal/g Protein/total kcal Carbohydrate/total kcal Fat/total kcal
g
kcal
47.25 16.0 29.4
189.0 64.0 117.6
2.4
0.6 2.0 1.0 3.5
18.0 3.95 1.65
0.2
0.2 0.8
100.0
397.6
0.05
3.98 0.302 0.636 0.045
Gavage Ethanol (95% USP) was mixed with Sustacal (Mead Johnson Nutritionals, Evansville, IN) to a concentration of 20% and administered (4 g/kg body wt) once per day in the morning, five days per week, Monday through Friday. Control animals were also gavaged by the same schedule with Sustacal supplemented with isocaloric sucrose in place of the ethanol. The volume gavaged did not exceed 0.7 ml/day even when the mice weighed >35 g, because greater volumes resulted in weight loss. Thus mice weighing >35 Ig received
2
60-
o E
40-
u c
3
20 -
60
20
70
Age (Weeks) Figure 1. Tendency to develop mammary tumors among isocalorically fed C3H7Ou mice in Expt 1, in which ethanol-treated mice (n = 15) consumed 12% ethanol in their drinking water and control mice (n = 15) consumed water alone. Mice of both groups consumed a semipurified solid diet described in Table 1.
248
Nutrition and Cancer 1992
40 -a—
Weight control
-•
Weight Ethanol
5 30-
Downloaded by [] at 18:02 30 December 2014
5
20
20
10
Age
30
40
(weeks)
Figure 2. Weight gain of mice fed semipunfied solid diet in Expt 1, in which mice received 12% ethanol in water or water alone. Values are means ± SEM.
(Figure 6). Mice consumed approximately 20 g/kg/day of ethanol. Weight gain in ethanol-fed animals was the same as in controls for 14 weeks and then decreased in the ethanol group in relation to the controls, despite continued pair feeding of the isocaloric diet (Figure 7). Discussion
Studies evaluating the influence of ethanol on experimental mammary tumorigenesis have been conflicting (10,11,14,15). In the experiments described here, ethanol did not enhance the rate of mammary tumor development in isocalorically fed mice despite a variety of methods of delivering ethanol to mice. In all the studies reported here, we used well-defined semipunfied solid or liquid diets and ensured isocaloric consumption. Whether mice consuming ethanol received it in the drinking water or in the semipurified liquid diet or were gavaged with ethanol and received the balance of their calories from solid diet, no substantial difference in the tendency to develop mammary tumors was noted that could be attributed to ethanol consumption. The lower weight gain with respect to energy intake and the statistically suggestive (p = 0.03) delay in mammary tumor development in ethanol-consuming mice in Experiment 1 may be attributable to reduced calorie utilization by these mice, representing a reduction in energy availability for growth and maintenance (16). This would suggest that ethanol interference with carbohydrate utilization in our first experiment may be responsible for the slight delay in the rate of tumor development in ethanol-fed animals, because they did not gain weight at the same rate as control animals despite the fact that the total calories consumed, including those contributed by ethanol, were approximately the same in the ethanol group as in the control group. If ethanol calories are excluded, then control animals consumed more solid diet calories than the ethanol group. Thus it is possible that ethanol in this study was acting
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