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Serum cholesterol and 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis
Cancer Letters, 14 (1981) 273-278 Elmvier/North-Holland Scientific PublishersLtd.
SERUM CHOLESTEROL AND 7,12_DIMETHYLBENZ[a] INDUCED MAMMARY CARCINOGENESIS
213
ANTHRACENE-
DAVID M. KLURFELD and DAVID KRITCHEVSKY The Wietar Institute of Anatomy 19104-4268 (U.S.A.)
and Biology,
36th Street at Spruce, Philadelphia,
PA
(Received 6 July 1981) (Revised version received 28 August 1981) (Accepted 28 August 1981)
SUMMARY
Diet-induced changes in serum cholesterol levels and their relationship to mammary carcinogenesis initiated by 7,12dimethylbenz[a] anthracene (DMBA) were studied in female Sprague-Dawley rats. DMBA was given to rats subjected to 3 dietary treatments: (1) a semipurified, cholesterolfree diet (SP); (2) the same diet with 1.5% cholesterol and 0.5% bile salts added (CB); (3) diet CB, until administration of DMBA and then switched to diet SP. Tumor yield per rat was increased in rats fed diet CB, but incidence and tumor size were similar among all 3 groups. Rats maintained on diet SP alone had a higher percentage of histologically benign tumors. Hypercholesterolemia of dietary origin appears to enhance slightly chemical carcinogenesis in this model.
INTRODUCTION
The relationship of serum cholesterol to the incidence of cancer in various organs is a matter of considerable interest. Because of the epidemiologic association of coronary heart disease with cancers of the breast and bowel or leukemia, it has been suggested that cholesterol may be involved in the pathogenesis of these diseases [15]. However, the evidence linking serum cholesterol and cancer is, at best, equivocal. One epidemiologic investigation found a negative association for cancer of several sites, including breast, and serum cholesterol concentrations all of which were determined 12-14 years previously [ 91. An inverse association between serum cholesterol levels and incidence of cancer of the colon and other sites was found for men, but not women, in the Framingham study [18]. Total cancer mortality among both male and female New Zealand Maoris was inversely correlated with serum cholesterol concentrations [ 11. A recent study of women with 0304-3836/81/0000+000/$02.76 o 1981 Elsevier/North-HollandScientific PublishersLtd.
274
breast cancer revealed that those patients with higher than medium serum cholesterol levels had reduced survival. A significant decrease in the survival rate among women with pre-operative body weights above the median was also observed. High serum cholesterol had a highly significant potentiating effect on the weight bias for 5-year survival [ 171. This influence of serum cholesterol levels on survival appears to contradict the findings for incidence of breast cancer in the epidemiologic studies [ 1,9,18] . Because serum cholesterol levels in both humans and laboratory animals are influenced by ingestion of fat and animal protein [ll],it is of interest that the international differences in breast cancer mortality correlate strongly with intake of fat [3,13] and animal protein [5,6]. However, it has not been possible to determine whether the associations of breast cancer mortality with dietary fat and animal protein are independent of one another [ 61. The data from human studies linking serum cholesterol levels with susceptibility to breast cancer indicated to us the need for animal studies in which other aspects of the diet could be controlled. This report describes an experiment in which rats with differing serum cholesterol levels were given a mammary carcinogen and subsequently, tumor formation was studied. MATERIALS
AND METHODS
Female Sprague-Dawley rats (Charles River Breeding Farm, Wilmington, MA) were received at 22 days of age. Thirty rats were immediately placed on a semipurified diet (SP) composed of: 47.0% dextrose; 21.7% casein; 0.3% DL-methionine; 10.0% cellulose; 5.0% Bernhart-Tomarelli salt mix; 1.0% AIN vitamin mix; 5.0% corn oil; 10.0% coconut oil. Another 60 rats were fed the above diet with 1.5% cholesterol and 0.5% bile salts (approx. 50% sodium cholate and 50% sodium deoxycholate) added at the expense of dextrose (CB). Food and water were provided ad libitum. At 50 days of age, each rat was given a single oral dose of 5 mg of DMBA (Eastman Kodak Co., Rochester, NY) dissolved in 0.5 ml corn oil, From 2 days before until 2 days after administration of the DMBA, all rats were fed the cholesterol-free diet so there would be no dietary effect on absorption of the carcinogen from the intestinal lumen. At this time, 30 of the animals fed the CB diet were continued on it while the other 30 were switched to the SP diet in order to lower their serum cholesterol levels. The animals were weighed monthly, palpated weekly for signs of tumor formation, and killed 4 months after the carcinogen was given. At necropsy, blood was obtained from 6 animals in each group for determination of serum cholesterol [ 141. Tumors were excised, weighed, measured, fixed in 10% buffered formalin and sections taken for histologic study. Histologic classification was determined according to criteria published by the IARC [ 191. Tumor volume was calculated by measuring each tumor in 3 perpendicular dimensions and multiplying these values.
275
Statistical analyses were performed using l- and 2-way analyses of variance for body weights and terminal results. An independent t-test was employed to analyze cholesterol levels at the time of carcinogen dosage and the chi-square test was used for statistical analysis of tumor yield [ 201. All data are given as mean + S.E.M. RESULTS
There were no significant differences in body weight due to diet or carcinogen administration throughout the course of the study. Terminal average weights of the 3 groups given DMBA were: SP, 356 f 7 g; CB, 336 f 6 g; CBSP, 350 f 9 g. Mean weights of the 3 dietary groups not given carcinogenwere: SP, 349 f 15 g; CB, 384 + 25 g; CB-SP, 335 + 20 g. Serum cholesterol levels at the time of DMBA dosing were 99 + 8 mg/lOO ml in the group fed the SP diet and 323 + 42 mg/lOO ml in the rats fed the CB diet (P < 0.001). Terminal cholesterol levels were 122 + 22 in group SP, 485 + 43 in group CB and 138 + 15 in the group switched from diet CB to SP following administration of the carcinogen (P < 0.001). Tumor incidence was similar in all 3 groups (69-73s) (Table 1). The number of tumors per tumor-bearing rat was 4.5 in the hypercholesterolemic rats and only 3.2 and 3.5 in the groups fed the SP diet throughout and after DMBA administration, respectively. Although these differences were not statistically significant, the total tumor yield was significantly greater in group CB than in the other 2 groups (P < 0.05). Both tumor volume and weight were similar among all 3 groups. The latency period of tumor appearance was 2 weeks longer in group SP than the other 2 groups. Group CB consistently had at least twice as many palpable tumors as group SP, while group CB-SP was intermediate in occurrence (see Fig. 1).
TABLE 1 TUMOR CHARACTERISTICS REGIMENS
FROM DMBA-TREATED
RATS FED THREE DIETARY
Dietary treatment* .~
Number of rats No. of tumor-bearing rats (96) Total no. of mammary tumors Tumors/tumor-bearing rat Tumor volume ( cm3) Tumor weight (g) Wee text for description
of diets.
SP
CB
CEs-SP
29 20 (69%) 63 3.2 f 0.5 2.66 f 0.84 1.8 * 0.5
30 22 (73%) 98 4.5 * 0.7 2.38 * 0.62 1.7 f 0.4
28 20 (71%) 69 3.5 f 0.7 2.52 f 0.60 2.0 f 0.5
276
50 45
/
,J
0
40 z p z
35 30
B a
25
0-O-Q
# 3> 2
20 15 10
WEEKS
Fig. 1. Cumulative incidence of palpable mammary tumors following -.);cB-sP(+~*~.*+). DMBA. Groups: SP (o);CB(.----
administration
of
Histologically, 95% of the mammary tumors were adenocarcinomas. In group SP, 14% of the tumors were classified as fibroadenoma compared to 1% in group CB and 2% in group CB-SP (P < 0.001). In rats fed diet CB, many tumors contained numerous cholesterol clefts and clusters of foam cell macrophages. A few tumors of other types were observed. Two rats in group SP had lymphocytic leukemia or lymphosarcoma involving the liver and spleen. Among rats fed diet CB, a small number of other tumors were found, including a nephroblastoma, a renal adenocarcinoma, a squamous cell carcinoma of the skin and a mammary adenoacanthoma. Because there were so few, it could not be determined if these other tumors were induced by DMBA or if the diets played any role in their development. DISCUSSION
The data suggest that diet-induced hypercholesterolemia exerts a slight enhancing effect on DMBA-induced mammary carcinogenesis. Reducing serum cholesterol, as in group CB-SP, reduces tumor formation to the level seen in the SP group. Szepsenwol [ 161 reported that addition of cholesterol plus lard to a commercial rodent diet enhanced the incidence of spontaneous mammary
277
and pulmonary adenocarcinomas in mice, but when the diet was supplemented with cholesterol alone, mammary tumors rarely developed. Slight, but significant protection against ultraviolet light-induced skin carcinogenesis is afforded mice by diet-induced hypercholesterolemia [ 21. Enrichment of lymphoma cells with cholesterol in vitro led to inhibition of tumor growth and increased host survival following transplantation into mice [ 81. Those results led the investigators to conclude that the cholesterol/phospholipid ratio of plasma membranes contributed to the bioregulation of growth in leukemic cells. This conclusion has been challenged by Chen et al. [4]. Cholesterol may also affect the endocrine and immune systems, thereby influencing mammary tumor development [ 10,121. The level of fat used in the present experiment has been shown to promote DMBA-induced carcinogenesis significantly [7]. This may have masked an effect of the dietary cholesterol. However, feeding the same diets to F344 rats given a colon carcinogen resulted in significantly increased numbers of colonic carcinomas in the animals maintained on the high cholesterol diet, although serum cholesterol levels actually fell in this other strain of rats during the study period (Klurfeld et al., unpublished). Further studies in laboratory animals are necessary to determine if differences in serum cholesterol concentrations have any relationship to carcinogenesis in specific organs. ACKNOWLEDGEMENTS
This work was supported, in part, by a Research Career Award (HL0734) from the National Institutes of Health and by grants-in-aid from the Commonwealth of Pennsylvania, The National Dairy Council, the Marketing Association of German Agriculture and the W.W. Smith Charitable Trust. REFERENCES 1 Beaglehole, R., Foulkes, M.A., Prior, I.A.M. and Eyles, E.F. (1980) Cholesterol and mortality in New Zealand Maoris. Br. Med. J., 280, 285-287. 2 Black, H.S., Henderson, S.V., Kleinhans, C.M., Phelps, A.W. and Thornhy, J.I. (1979) Effect of dietary cholesterol in ultraviolet light carcinogenesis. Cancer Res., 39, 5022-5027. 3 Carroll, K.K. and Khor, H.T. (1975) Dietary fat in relation to tumorigenesis. Prog. Biochem. Pharmacol., 10, 308-353. 4 Chen, H.W., Kandutsch, A.A. and Heiniger, H.J. (1978) ‘Ihe role of cholesterol in malignancy. Prog. Exp. Tumor Res., 22,275-316. 5 Drasar, B.S. and Irving, D. (1973) Environmental factors and cancer of the colon and breast. Br. J. Cancer, 27,167-172. 6 Hems, G. (1978) The contributions of diet and childbearing to breast-cancer rates. Br. J. Cancer, 37,974-982. 7 Hopkins, G.J. and Carroll, K.K. (1979) Relationship between amount and type of dietary fat in promotion of mammary carcinogeneeis induced by 7 ,12_dimethylbenz[alanthracene. J. Natl. Cancer Inst., 62,1009-1012. 8 Inbar, M. and Shinitzky, M. (1974) Increase of cholesterol levele in the surface
278
9
10
11 12
13 14
membrane of lymphoma cells and its inhibitory effect on ascites tumor development. Proc. Natl. Acad. Sci. U.S.A., 71, 2128-2130. Kark, J.D., Smith, A.H. and Hames, C.G. (1980) The relationship of serum cholesterol to the incidence of cancer in Evans County, Georgia. J. Chronic Dis., 33, 311-322. Klurfeld, D.M., Allison, M.J., Gerszten, E. and Dalton, H.P. (1979) Alterations of host defenses paralleling cholesterol-induced atherogenesis. II. Immunologic studies of rabbits. J. Med., 10,49-64. Kritchevsky, D. (1976) Diet and atherosclerosis. Am. J. Pathol., 84, 615-632. Kritchevsky, D. and Klurfeld, D.M. (1981) Fat and Cancer. In: Nutrition and Cancer: Etiology and Treatment, pp. 173-188. Editors: G.R. Newell and M.M. Ellison. Raven Press, New York. Lea, A.J. (1966) Dietary factors associated with death-rates from certain neoplasms in man. Lancet, 2, 332-333. Roschlau, P., Remt, E. and Gruber, W. (1974) Enzymatische bestimmung des gesamtcholesterins in serum. Z. Klin. Chem. Klin. B&hem., 12,403-407.
15 Rose, G., Blackbum, H., Keys, A., Taylor, H.L., Kannel, W.B., Paul, O., Reid, D.D. and Stamler, J. (1974) Colon cancer and blood-cholesterol. Lancet, 1, 181-183. 16 Szepsenwol, J. (1964) Carcinogenic effect of ether extract of whole egg, alcohol extract of egg yolk and powdered egg free of the ether extractable part in mice. Proc. Sot. Exp. Biol. Med., 116,1136-1139. 17 Tartter, P.I., Papatestas, A.E., Ioannovich, J., Mulvihill, M.N., Lesnick, G. and Aufses, A.H. (1981) Cholesterol and obesity as prognostic factors in breast cancer. Cancer, 47,2222-2227. 18 Williams, R.R., Sorlie, P.D., Feinleib, M., McNamara, P.M., Kannel, W.B. and Dawber, T.R. (1981) Cancer incidence by levels of cholesterol. J. Am. Med. Assoc., 245, 247-253. 19 Young, S. and Hallowes, R.C. (1973) Tumors of the mammary gland. In: Pathology of Tumors in Laboratory Animals, Vol. 1, Part 1, pp. 31-74. Editor: VS. Turusov. International Agency for Research on Cancer, Lyon. 20 Zar, J.H. (1974) Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, NJ.
SERUM CHOLESTEROL AND 7,12_DIMETHYLBENZ[a] INDUCED MAMMARY CARCINOGENESIS
213
ANTHRACENE-
DAVID M. KLURFELD and DAVID KRITCHEVSKY The Wietar Institute of Anatomy 19104-4268 (U.S.A.)
and Biology,
36th Street at Spruce, Philadelphia,
PA
(Received 6 July 1981) (Revised version received 28 August 1981) (Accepted 28 August 1981)
SUMMARY
Diet-induced changes in serum cholesterol levels and their relationship to mammary carcinogenesis initiated by 7,12dimethylbenz[a] anthracene (DMBA) were studied in female Sprague-Dawley rats. DMBA was given to rats subjected to 3 dietary treatments: (1) a semipurified, cholesterolfree diet (SP); (2) the same diet with 1.5% cholesterol and 0.5% bile salts added (CB); (3) diet CB, until administration of DMBA and then switched to diet SP. Tumor yield per rat was increased in rats fed diet CB, but incidence and tumor size were similar among all 3 groups. Rats maintained on diet SP alone had a higher percentage of histologically benign tumors. Hypercholesterolemia of dietary origin appears to enhance slightly chemical carcinogenesis in this model.
INTRODUCTION
The relationship of serum cholesterol to the incidence of cancer in various organs is a matter of considerable interest. Because of the epidemiologic association of coronary heart disease with cancers of the breast and bowel or leukemia, it has been suggested that cholesterol may be involved in the pathogenesis of these diseases [15]. However, the evidence linking serum cholesterol and cancer is, at best, equivocal. One epidemiologic investigation found a negative association for cancer of several sites, including breast, and serum cholesterol concentrations all of which were determined 12-14 years previously [ 91. An inverse association between serum cholesterol levels and incidence of cancer of the colon and other sites was found for men, but not women, in the Framingham study [18]. Total cancer mortality among both male and female New Zealand Maoris was inversely correlated with serum cholesterol concentrations [ 11. A recent study of women with 0304-3836/81/0000+000/$02.76 o 1981 Elsevier/North-HollandScientific PublishersLtd.
274
breast cancer revealed that those patients with higher than medium serum cholesterol levels had reduced survival. A significant decrease in the survival rate among women with pre-operative body weights above the median was also observed. High serum cholesterol had a highly significant potentiating effect on the weight bias for 5-year survival [ 171. This influence of serum cholesterol levels on survival appears to contradict the findings for incidence of breast cancer in the epidemiologic studies [ 1,9,18] . Because serum cholesterol levels in both humans and laboratory animals are influenced by ingestion of fat and animal protein [ll],it is of interest that the international differences in breast cancer mortality correlate strongly with intake of fat [3,13] and animal protein [5,6]. However, it has not been possible to determine whether the associations of breast cancer mortality with dietary fat and animal protein are independent of one another [ 61. The data from human studies linking serum cholesterol levels with susceptibility to breast cancer indicated to us the need for animal studies in which other aspects of the diet could be controlled. This report describes an experiment in which rats with differing serum cholesterol levels were given a mammary carcinogen and subsequently, tumor formation was studied. MATERIALS
AND METHODS
Female Sprague-Dawley rats (Charles River Breeding Farm, Wilmington, MA) were received at 22 days of age. Thirty rats were immediately placed on a semipurified diet (SP) composed of: 47.0% dextrose; 21.7% casein; 0.3% DL-methionine; 10.0% cellulose; 5.0% Bernhart-Tomarelli salt mix; 1.0% AIN vitamin mix; 5.0% corn oil; 10.0% coconut oil. Another 60 rats were fed the above diet with 1.5% cholesterol and 0.5% bile salts (approx. 50% sodium cholate and 50% sodium deoxycholate) added at the expense of dextrose (CB). Food and water were provided ad libitum. At 50 days of age, each rat was given a single oral dose of 5 mg of DMBA (Eastman Kodak Co., Rochester, NY) dissolved in 0.5 ml corn oil, From 2 days before until 2 days after administration of the DMBA, all rats were fed the cholesterol-free diet so there would be no dietary effect on absorption of the carcinogen from the intestinal lumen. At this time, 30 of the animals fed the CB diet were continued on it while the other 30 were switched to the SP diet in order to lower their serum cholesterol levels. The animals were weighed monthly, palpated weekly for signs of tumor formation, and killed 4 months after the carcinogen was given. At necropsy, blood was obtained from 6 animals in each group for determination of serum cholesterol [ 141. Tumors were excised, weighed, measured, fixed in 10% buffered formalin and sections taken for histologic study. Histologic classification was determined according to criteria published by the IARC [ 191. Tumor volume was calculated by measuring each tumor in 3 perpendicular dimensions and multiplying these values.
275
Statistical analyses were performed using l- and 2-way analyses of variance for body weights and terminal results. An independent t-test was employed to analyze cholesterol levels at the time of carcinogen dosage and the chi-square test was used for statistical analysis of tumor yield [ 201. All data are given as mean + S.E.M. RESULTS
There were no significant differences in body weight due to diet or carcinogen administration throughout the course of the study. Terminal average weights of the 3 groups given DMBA were: SP, 356 f 7 g; CB, 336 f 6 g; CBSP, 350 f 9 g. Mean weights of the 3 dietary groups not given carcinogenwere: SP, 349 f 15 g; CB, 384 + 25 g; CB-SP, 335 + 20 g. Serum cholesterol levels at the time of DMBA dosing were 99 + 8 mg/lOO ml in the group fed the SP diet and 323 + 42 mg/lOO ml in the rats fed the CB diet (P < 0.001). Terminal cholesterol levels were 122 + 22 in group SP, 485 + 43 in group CB and 138 + 15 in the group switched from diet CB to SP following administration of the carcinogen (P < 0.001). Tumor incidence was similar in all 3 groups (69-73s) (Table 1). The number of tumors per tumor-bearing rat was 4.5 in the hypercholesterolemic rats and only 3.2 and 3.5 in the groups fed the SP diet throughout and after DMBA administration, respectively. Although these differences were not statistically significant, the total tumor yield was significantly greater in group CB than in the other 2 groups (P < 0.05). Both tumor volume and weight were similar among all 3 groups. The latency period of tumor appearance was 2 weeks longer in group SP than the other 2 groups. Group CB consistently had at least twice as many palpable tumors as group SP, while group CB-SP was intermediate in occurrence (see Fig. 1).
TABLE 1 TUMOR CHARACTERISTICS REGIMENS
FROM DMBA-TREATED
RATS FED THREE DIETARY
Dietary treatment* .~
Number of rats No. of tumor-bearing rats (96) Total no. of mammary tumors Tumors/tumor-bearing rat Tumor volume ( cm3) Tumor weight (g) Wee text for description
of diets.
SP
CB
CEs-SP
29 20 (69%) 63 3.2 f 0.5 2.66 f 0.84 1.8 * 0.5
30 22 (73%) 98 4.5 * 0.7 2.38 * 0.62 1.7 f 0.4
28 20 (71%) 69 3.5 f 0.7 2.52 f 0.60 2.0 f 0.5
276
50 45
/
,J
0
40 z p z
35 30
B a
25
0-O-Q
# 3> 2
20 15 10
WEEKS
Fig. 1. Cumulative incidence of palpable mammary tumors following -.);cB-sP(+~*~.*+). DMBA. Groups: SP (o);CB(.----
administration
of
Histologically, 95% of the mammary tumors were adenocarcinomas. In group SP, 14% of the tumors were classified as fibroadenoma compared to 1% in group CB and 2% in group CB-SP (P < 0.001). In rats fed diet CB, many tumors contained numerous cholesterol clefts and clusters of foam cell macrophages. A few tumors of other types were observed. Two rats in group SP had lymphocytic leukemia or lymphosarcoma involving the liver and spleen. Among rats fed diet CB, a small number of other tumors were found, including a nephroblastoma, a renal adenocarcinoma, a squamous cell carcinoma of the skin and a mammary adenoacanthoma. Because there were so few, it could not be determined if these other tumors were induced by DMBA or if the diets played any role in their development. DISCUSSION
The data suggest that diet-induced hypercholesterolemia exerts a slight enhancing effect on DMBA-induced mammary carcinogenesis. Reducing serum cholesterol, as in group CB-SP, reduces tumor formation to the level seen in the SP group. Szepsenwol [ 161 reported that addition of cholesterol plus lard to a commercial rodent diet enhanced the incidence of spontaneous mammary
277
and pulmonary adenocarcinomas in mice, but when the diet was supplemented with cholesterol alone, mammary tumors rarely developed. Slight, but significant protection against ultraviolet light-induced skin carcinogenesis is afforded mice by diet-induced hypercholesterolemia [ 21. Enrichment of lymphoma cells with cholesterol in vitro led to inhibition of tumor growth and increased host survival following transplantation into mice [ 81. Those results led the investigators to conclude that the cholesterol/phospholipid ratio of plasma membranes contributed to the bioregulation of growth in leukemic cells. This conclusion has been challenged by Chen et al. [4]. Cholesterol may also affect the endocrine and immune systems, thereby influencing mammary tumor development [ 10,121. The level of fat used in the present experiment has been shown to promote DMBA-induced carcinogenesis significantly [7]. This may have masked an effect of the dietary cholesterol. However, feeding the same diets to F344 rats given a colon carcinogen resulted in significantly increased numbers of colonic carcinomas in the animals maintained on the high cholesterol diet, although serum cholesterol levels actually fell in this other strain of rats during the study period (Klurfeld et al., unpublished). Further studies in laboratory animals are necessary to determine if differences in serum cholesterol concentrations have any relationship to carcinogenesis in specific organs. ACKNOWLEDGEMENTS
This work was supported, in part, by a Research Career Award (HL0734) from the National Institutes of Health and by grants-in-aid from the Commonwealth of Pennsylvania, The National Dairy Council, the Marketing Association of German Agriculture and the W.W. Smith Charitable Trust. REFERENCES 1 Beaglehole, R., Foulkes, M.A., Prior, I.A.M. and Eyles, E.F. (1980) Cholesterol and mortality in New Zealand Maoris. Br. Med. J., 280, 285-287. 2 Black, H.S., Henderson, S.V., Kleinhans, C.M., Phelps, A.W. and Thornhy, J.I. (1979) Effect of dietary cholesterol in ultraviolet light carcinogenesis. Cancer Res., 39, 5022-5027. 3 Carroll, K.K. and Khor, H.T. (1975) Dietary fat in relation to tumorigenesis. Prog. Biochem. Pharmacol., 10, 308-353. 4 Chen, H.W., Kandutsch, A.A. and Heiniger, H.J. (1978) ‘Ihe role of cholesterol in malignancy. Prog. Exp. Tumor Res., 22,275-316. 5 Drasar, B.S. and Irving, D. (1973) Environmental factors and cancer of the colon and breast. Br. J. Cancer, 27,167-172. 6 Hems, G. (1978) The contributions of diet and childbearing to breast-cancer rates. Br. J. Cancer, 37,974-982. 7 Hopkins, G.J. and Carroll, K.K. (1979) Relationship between amount and type of dietary fat in promotion of mammary carcinogeneeis induced by 7 ,12_dimethylbenz[alanthracene. J. Natl. Cancer Inst., 62,1009-1012. 8 Inbar, M. and Shinitzky, M. (1974) Increase of cholesterol levele in the surface
278
9
10
11 12
13 14
membrane of lymphoma cells and its inhibitory effect on ascites tumor development. Proc. Natl. Acad. Sci. U.S.A., 71, 2128-2130. Kark, J.D., Smith, A.H. and Hames, C.G. (1980) The relationship of serum cholesterol to the incidence of cancer in Evans County, Georgia. J. Chronic Dis., 33, 311-322. Klurfeld, D.M., Allison, M.J., Gerszten, E. and Dalton, H.P. (1979) Alterations of host defenses paralleling cholesterol-induced atherogenesis. II. Immunologic studies of rabbits. J. Med., 10,49-64. Kritchevsky, D. (1976) Diet and atherosclerosis. Am. J. Pathol., 84, 615-632. Kritchevsky, D. and Klurfeld, D.M. (1981) Fat and Cancer. In: Nutrition and Cancer: Etiology and Treatment, pp. 173-188. Editors: G.R. Newell and M.M. Ellison. Raven Press, New York. Lea, A.J. (1966) Dietary factors associated with death-rates from certain neoplasms in man. Lancet, 2, 332-333. Roschlau, P., Remt, E. and Gruber, W. (1974) Enzymatische bestimmung des gesamtcholesterins in serum. Z. Klin. Chem. Klin. B&hem., 12,403-407.
15 Rose, G., Blackbum, H., Keys, A., Taylor, H.L., Kannel, W.B., Paul, O., Reid, D.D. and Stamler, J. (1974) Colon cancer and blood-cholesterol. Lancet, 1, 181-183. 16 Szepsenwol, J. (1964) Carcinogenic effect of ether extract of whole egg, alcohol extract of egg yolk and powdered egg free of the ether extractable part in mice. Proc. Sot. Exp. Biol. Med., 116,1136-1139. 17 Tartter, P.I., Papatestas, A.E., Ioannovich, J., Mulvihill, M.N., Lesnick, G. and Aufses, A.H. (1981) Cholesterol and obesity as prognostic factors in breast cancer. Cancer, 47,2222-2227. 18 Williams, R.R., Sorlie, P.D., Feinleib, M., McNamara, P.M., Kannel, W.B. and Dawber, T.R. (1981) Cancer incidence by levels of cholesterol. J. Am. Med. Assoc., 245, 247-253. 19 Young, S. and Hallowes, R.C. (1973) Tumors of the mammary gland. In: Pathology of Tumors in Laboratory Animals, Vol. 1, Part 1, pp. 31-74. Editor: VS. Turusov. International Agency for Research on Cancer, Lyon. 20 Zar, J.H. (1974) Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, NJ.