Jr, Hellman S, Rosenberg SA, eds), vol 3, No. 10. Philadelphia: Lippincott, 1989, pp 1-12 (4) ALLEY MC, SCUDIERO DA, MONKS A, ET AL: Feasibility of drug screening
with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res 48:589-601, 1988 (5) SCUDIERO DA, SHOEMAKER RH, PAUU. KD, ET AL: Evaluation of a soluble
tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 48:4827^*833, 1988 (6) SKEHAN
P, STORENG
R, SCUCHERO DA,
ET AL: New
colorimetric
cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82:1107-1112,1990 (7) STINSON SF, ALLEY MC, KENNEY S, ET AL: Morphologic characterization
of human cell carcinoma cell lines. Presented at the annual meeting of the American Association for Cancer Research, San Francisco, Calif, May 2427, 1989 (8) SKEHAN P: On the normality of growth dynamics of neoplasms in vivo: Data base analysis. Growth 50:496-515, 1986 (9) VisncA DT, SKEHAN P, SCUDIERO DA, ET AL: Tetrazolium-based assays for cellular viability: A critical examination of parameters which affect formazan production. Cancer Res. In press
(10) VISTICA DT, SCUDIERO DA, SKEHAN P, ET AL: New carbon dioxide-independent basal growth medium for culture of diverse tumor and nontumor cells of human and nonhuman origin. J Nat] Cancer Inst 82:1055-1061, 1990 (//) RUBINSTEIN LV, PAULL KD, SHOEMAKER RH, ET AL: Comparison of in vitro anticancer drug screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines. J Natl Cancer Inst 82:1113-1118,1990 (12) AD HOC REVIEW COMMITTEE: Proceedings for National Cancer Institute In Vitro/In Vivo Disease-Oriented Screening Project. Bethesda, Md: NIH, Nov 13-15,1989 (13) Reviewers Report Progress in New Drug Prescreen System Development. Cancer Lett 15:1-5, 1989 (14) BOYD MR, PAULL KD, RUBENSTEIN LR: Data display and analysis strategies for the NCI disease-oriented in vitro antitumor drug screen. In Proceedings of the 22nd Annual Detroit Oncology Symposium on Anticancer Drug Discovery and Development (Valeriote FA, Corbett T, Baker L, eds). Detroit, April 25-27, 1990 (15) PAULL KD, SHOEMAKER RH, HODES L, ET AL: Display and analysis of patterns of differential activity of drugs against human tumor cell lines. J Natl Cancer Inst 81:1088-1092, 1989
Zora Djuric * Lance K. Heilbrun, Bruce A. Reading, Allison Boomer, Frederick A. Valeriote, Silvana Martino
Fat in the diet has been associated with increased breast cancer risk. In this study, blood samples were obtained from 21 women at high risk for breast cancer who had been randomly assigned to either a nonintervention diet or a low-fat diet. Oxidative damage was examined in the DNA from nucleated peripheral blood cells. The levels of oxidized thymine, specifically 5-hydroxymethyluracil, were threefold higher in the nonintervention diet group than in the low-fat diet group. Without regard to diet arm, there also was a significant linear relationship between daily total fat intake and 5-hydroxymethyluracil level. These results suggest that oxidative damage to DNA may be a marker of dietary fat intake. In addition, oxidative DNA damage may be a mechanistic link between fat in the diet and cancer risk, since such damage is associated with the process of tumor promotion. [J Natl Cancer Inst 83:766-769,1991]
Epidemiological studies have suggested an association between increased fat intake and breast cancer risk {12). In an effort to modulate breast cancer risk in women at high risk for breast cancer, we initiated an intervention study that randomly assigned high-risk women to either a nonintervention diet or a 766
low-fat diet. The diet of each woman is being closely monitored, and potential markers of fat intake and cancer risk are being established. One intermediate marker of breast cancer risk may be oxidative damage to the DNA. Such damage may play a role in tumor promotion (J) and can be elicited by numerous endogenous processes, including lipid peroxidation (4). When DNA is exposed to oxidants, thymine residues appear to be highly susceptible to oxidation (5,6). Of the thymine oxidation products,
Received October 1, 1990; revised February 28, 1991; accepted March 6, 1991. Supported by the Wayne State University Ben Kastle Trust for Cancer Research and by Public Health Service grant CA-22453 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. The GC/MS data were obtained at the Michigan State University Mass Spectrometry Facility (East Lansing, Mich), which is supported in part by grant RR-00480 from the Division of Research Resources, National Cancer Institute, National Institutes of Health. Presented in part at the 5th Biennial Meeting of the Society for Free-Radical Research, Pasadena, Calif, November 14-18, 1990. Department of Internal Medicine, Wayne State University, Detroit, Mich. We thank Maria Alonso for blood sample collection. 'Correspondence to: Zora Djuric, PhD, Oncology Division, Wayne State University, PO Box 02188, Detroit, MI 48201.
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at New York University on October 18, 2014
Effects of a Low-Fat Diet on Levels of Oxidative Damage to DNA in Human Peripheral Nucleated Blood Cells
5-hydroxymethyluracil is relatively more stable than thymine glycol (7). In addition, 5-hydroxymethyluracil may be a mutagenic lesion (8), while thymine glycol is unlikely to be mutagenic (9). -We quantified the levels of 5-hydroxymethyluracil in the DNA from nucleated peripheral blood cells of 21 women in our dietary intervention program. The data indicated that 5-hydroxymethyluracil levels can be decreased by a low-fat diet.
Patients, Materials, and Methods
Vol. 83, No. 11, June 5, 1991
Results The level of fat intake was calculated from dietary records obtained at the same time the blood was drawn for analysis of DNA damage. At the time the blood was drawn, the 21 women had been in the dietary intervention program between 3 and 24 months. The mean total fat intake in the low-fat group was significantly lower than that in the nonintervention group (32.5 vs 57.0 g/day; Table 1). Similarly, the mean intakes of saturated fat, monounsaturated fat, and polyunsaturated fat in the low-fat group were decreased to 52%-58% of that in the nonintervention diet group. Statistical interpretation of the differences in mean intakes of specific fatty acids was not warranted in this preliminary report of DNA damage levels, especially in view of the study design which had the single purpose of lowering total fat intake. The mean level of 5-hydroxymethyluracil in the DNA of peripheral nucleated blood cells from women on the low-fat diet was significantly lower than that for the nonintervention diet group (2.99 vs 9.29 5-hydroxymethyluracil/lO4 thymine; Table 1). Seven other variables were compared by diet arm: age, body weight, percent body fat, and daily intake of calories, vitamin C, vitamin E, and fi-carotene. Mean percent body fat differed significantly (P = .013) by diet arm: 26.8% vs 32.4% for women on the low-fat and nonintervention diet arms, respectively. None of the remaining six variables were statistically different between the two diet groups (P>.21 in each case). The mean DNA damage level for premenopausal women was slightly lower than that for postmenopausal women. This result was consistent for each diet group and overall for the 21 women, but none of these differences were statistically significant. Among the 21 women, there was a significant linear regression relationship of daily total fat intake with the square root of
Downloaded from http://jnci.oxfordjournals.org/ at New York University on October 18, 2014
Patients. Women aged 18-67 years were eligible for the dietary intervention program if they satisfied one or more of the following criteria: (a) at least one first-degree relative with a diagnosis of breast cancer, (b) a mammogram classified as P2 or DY according to the Wolfe classification system {10), and (c) atypia or florid papillomatosis detected on breast biopsy. In addition, dietary fat intake upon entry to the program had to be at least 30% of the total caloric intake. The blood was drawn after a 14-hour overnight fast. The 15 premenopausal women were between days 17 and 24 of the menstrual cycle. All subjects gave their informed consent for participation in the study. Of the women in the program, a consecutive series of 21, aged 19-57 years, were selected for analysis of oxidative damage to DNA. Dietary intervention. The women were randomly assigned to a nonintervention diet or a low-fat diet after stratification into three age groups. The women on the nonintervention diet followed their own usual diet. The women in the low-fat diet group were instructed by a dietitian on how to follow the Low Fat Eating Plan developed by the American Health Foundation and the University of Minnesota Nutrition Coordinating Center (77). This diet has a target of 15% of calories from fat. The 3-day food records and 24-hour recall data of both groups were collected on site by a registered dietitian, and the diet was analyzed for nutritional content by staff at the University of Minnesota (12). Blood samples. All blood samples were drawn into heparinized tubes after a 14-hour fast. The blood was refrigerated immediately and subsequently stored at -70°C until analysis. Nuclei were prepared from 5- to 10-mL samples of heparinized blood by the method of Ciulla et al (13), and the DNA was isolated using organic extractions (14). The DNA was hydrolyzed, derivatized, and analyzed by gas chromatography with mass spectral detection, as described by Fuciarelli et al (5) with the following modifications. The amount of thymine in each sample was determined using thymine-d4 as an internal standard. This procedure controlled for the amount of DNA injected onto the column and served as a monitor of DNA hydrolysis efficiency. The amount of 5-hydroxymethyluracil in each sample was determined using 5-hydroxymethyluracil-2-'3C,5-^2,6-
with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res 48:589-601, 1988 (5) SCUDIERO DA, SHOEMAKER RH, PAUU. KD, ET AL: Evaluation of a soluble
tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 48:4827^*833, 1988 (6) SKEHAN
P, STORENG
R, SCUCHERO DA,
ET AL: New
colorimetric
cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82:1107-1112,1990 (7) STINSON SF, ALLEY MC, KENNEY S, ET AL: Morphologic characterization
of human cell carcinoma cell lines. Presented at the annual meeting of the American Association for Cancer Research, San Francisco, Calif, May 2427, 1989 (8) SKEHAN P: On the normality of growth dynamics of neoplasms in vivo: Data base analysis. Growth 50:496-515, 1986 (9) VisncA DT, SKEHAN P, SCUDIERO DA, ET AL: Tetrazolium-based assays for cellular viability: A critical examination of parameters which affect formazan production. Cancer Res. In press
(10) VISTICA DT, SCUDIERO DA, SKEHAN P, ET AL: New carbon dioxide-independent basal growth medium for culture of diverse tumor and nontumor cells of human and nonhuman origin. J Nat] Cancer Inst 82:1055-1061, 1990 (//) RUBINSTEIN LV, PAULL KD, SHOEMAKER RH, ET AL: Comparison of in vitro anticancer drug screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines. J Natl Cancer Inst 82:1113-1118,1990 (12) AD HOC REVIEW COMMITTEE: Proceedings for National Cancer Institute In Vitro/In Vivo Disease-Oriented Screening Project. Bethesda, Md: NIH, Nov 13-15,1989 (13) Reviewers Report Progress in New Drug Prescreen System Development. Cancer Lett 15:1-5, 1989 (14) BOYD MR, PAULL KD, RUBENSTEIN LR: Data display and analysis strategies for the NCI disease-oriented in vitro antitumor drug screen. In Proceedings of the 22nd Annual Detroit Oncology Symposium on Anticancer Drug Discovery and Development (Valeriote FA, Corbett T, Baker L, eds). Detroit, April 25-27, 1990 (15) PAULL KD, SHOEMAKER RH, HODES L, ET AL: Display and analysis of patterns of differential activity of drugs against human tumor cell lines. J Natl Cancer Inst 81:1088-1092, 1989
Zora Djuric * Lance K. Heilbrun, Bruce A. Reading, Allison Boomer, Frederick A. Valeriote, Silvana Martino
Fat in the diet has been associated with increased breast cancer risk. In this study, blood samples were obtained from 21 women at high risk for breast cancer who had been randomly assigned to either a nonintervention diet or a low-fat diet. Oxidative damage was examined in the DNA from nucleated peripheral blood cells. The levels of oxidized thymine, specifically 5-hydroxymethyluracil, were threefold higher in the nonintervention diet group than in the low-fat diet group. Without regard to diet arm, there also was a significant linear relationship between daily total fat intake and 5-hydroxymethyluracil level. These results suggest that oxidative damage to DNA may be a marker of dietary fat intake. In addition, oxidative DNA damage may be a mechanistic link between fat in the diet and cancer risk, since such damage is associated with the process of tumor promotion. [J Natl Cancer Inst 83:766-769,1991]
Epidemiological studies have suggested an association between increased fat intake and breast cancer risk {12). In an effort to modulate breast cancer risk in women at high risk for breast cancer, we initiated an intervention study that randomly assigned high-risk women to either a nonintervention diet or a 766
low-fat diet. The diet of each woman is being closely monitored, and potential markers of fat intake and cancer risk are being established. One intermediate marker of breast cancer risk may be oxidative damage to the DNA. Such damage may play a role in tumor promotion (J) and can be elicited by numerous endogenous processes, including lipid peroxidation (4). When DNA is exposed to oxidants, thymine residues appear to be highly susceptible to oxidation (5,6). Of the thymine oxidation products,
Received October 1, 1990; revised February 28, 1991; accepted March 6, 1991. Supported by the Wayne State University Ben Kastle Trust for Cancer Research and by Public Health Service grant CA-22453 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. The GC/MS data were obtained at the Michigan State University Mass Spectrometry Facility (East Lansing, Mich), which is supported in part by grant RR-00480 from the Division of Research Resources, National Cancer Institute, National Institutes of Health. Presented in part at the 5th Biennial Meeting of the Society for Free-Radical Research, Pasadena, Calif, November 14-18, 1990. Department of Internal Medicine, Wayne State University, Detroit, Mich. We thank Maria Alonso for blood sample collection. 'Correspondence to: Zora Djuric, PhD, Oncology Division, Wayne State University, PO Box 02188, Detroit, MI 48201.
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at New York University on October 18, 2014
Effects of a Low-Fat Diet on Levels of Oxidative Damage to DNA in Human Peripheral Nucleated Blood Cells
5-hydroxymethyluracil is relatively more stable than thymine glycol (7). In addition, 5-hydroxymethyluracil may be a mutagenic lesion (8), while thymine glycol is unlikely to be mutagenic (9). -We quantified the levels of 5-hydroxymethyluracil in the DNA from nucleated peripheral blood cells of 21 women in our dietary intervention program. The data indicated that 5-hydroxymethyluracil levels can be decreased by a low-fat diet.
Patients, Materials, and Methods
Vol. 83, No. 11, June 5, 1991
Results The level of fat intake was calculated from dietary records obtained at the same time the blood was drawn for analysis of DNA damage. At the time the blood was drawn, the 21 women had been in the dietary intervention program between 3 and 24 months. The mean total fat intake in the low-fat group was significantly lower than that in the nonintervention group (32.5 vs 57.0 g/day; Table 1). Similarly, the mean intakes of saturated fat, monounsaturated fat, and polyunsaturated fat in the low-fat group were decreased to 52%-58% of that in the nonintervention diet group. Statistical interpretation of the differences in mean intakes of specific fatty acids was not warranted in this preliminary report of DNA damage levels, especially in view of the study design which had the single purpose of lowering total fat intake. The mean level of 5-hydroxymethyluracil in the DNA of peripheral nucleated blood cells from women on the low-fat diet was significantly lower than that for the nonintervention diet group (2.99 vs 9.29 5-hydroxymethyluracil/lO4 thymine; Table 1). Seven other variables were compared by diet arm: age, body weight, percent body fat, and daily intake of calories, vitamin C, vitamin E, and fi-carotene. Mean percent body fat differed significantly (P = .013) by diet arm: 26.8% vs 32.4% for women on the low-fat and nonintervention diet arms, respectively. None of the remaining six variables were statistically different between the two diet groups (P>.21 in each case). The mean DNA damage level for premenopausal women was slightly lower than that for postmenopausal women. This result was consistent for each diet group and overall for the 21 women, but none of these differences were statistically significant. Among the 21 women, there was a significant linear regression relationship of daily total fat intake with the square root of
Downloaded from http://jnci.oxfordjournals.org/ at New York University on October 18, 2014
Patients. Women aged 18-67 years were eligible for the dietary intervention program if they satisfied one or more of the following criteria: (a) at least one first-degree relative with a diagnosis of breast cancer, (b) a mammogram classified as P2 or DY according to the Wolfe classification system {10), and (c) atypia or florid papillomatosis detected on breast biopsy. In addition, dietary fat intake upon entry to the program had to be at least 30% of the total caloric intake. The blood was drawn after a 14-hour overnight fast. The 15 premenopausal women were between days 17 and 24 of the menstrual cycle. All subjects gave their informed consent for participation in the study. Of the women in the program, a consecutive series of 21, aged 19-57 years, were selected for analysis of oxidative damage to DNA. Dietary intervention. The women were randomly assigned to a nonintervention diet or a low-fat diet after stratification into three age groups. The women on the nonintervention diet followed their own usual diet. The women in the low-fat diet group were instructed by a dietitian on how to follow the Low Fat Eating Plan developed by the American Health Foundation and the University of Minnesota Nutrition Coordinating Center (77). This diet has a target of 15% of calories from fat. The 3-day food records and 24-hour recall data of both groups were collected on site by a registered dietitian, and the diet was analyzed for nutritional content by staff at the University of Minnesota (12). Blood samples. All blood samples were drawn into heparinized tubes after a 14-hour fast. The blood was refrigerated immediately and subsequently stored at -70°C until analysis. Nuclei were prepared from 5- to 10-mL samples of heparinized blood by the method of Ciulla et al (13), and the DNA was isolated using organic extractions (14). The DNA was hydrolyzed, derivatized, and analyzed by gas chromatography with mass spectral detection, as described by Fuciarelli et al (5) with the following modifications. The amount of thymine in each sample was determined using thymine-d4 as an internal standard. This procedure controlled for the amount of DNA injected onto the column and served as a monitor of DNA hydrolysis efficiency. The amount of 5-hydroxymethyluracil in each sample was determined using 5-hydroxymethyluracil-2-'3C,5-^2,6-
