Diindolylmethane FAQs - "All About DIM"

Published Articles on DIM

Safer Estrogen with Phytonutrition
by Michael A. Zeligs, M.D.

Abstract
Phytonutrition encompasses the dietary use of micronutrients found in plants. Adequate intake of specific phytochemicals can increase adaptive responses regulating hormone metabolism and cell behavior. Cruciferous vegetables, such as cabbage, cauliflower, and broccoli, posses unique phytochemical constituents able to modify the metabolism of estrogen. The most active of these phytochemicals with regard to estrogen is the dietary indole, diindolylmethane. Supplemental use of diindolylmethane provides the basis for nutritional support to enhance the beneficial action and safety of estrogen. An optimal "estrogen balance" has implications for cancer prevention and successful aging in both women and men.

Click to view large diagram

Introduction: Dietary Ingredients Improve Estrogen Metabolism
Though discovered over ten years ago, the connection between plant-derived dietary ingredients and estrogen is just beginning to be appreciated. This connection has the power to explain much of the mystery of why people living in developed nations, but lacking dietary "phytonutrients", suffer disproportionately from the major hormone dependent cancers, colon cancer, and coronary heart disease.1 H. Leon Bradlow, Ph.D. and his group at the Strang Cancer Prevention Laboratory in New York were the first to establish the link between phytonutrients from cruciferous vegetables and estrogen metabolism. 2 They showed that supplemental use of a single cruciferous phytochemical can act to promote a dramatic and beneficial change in the metabolism of estrogen.3 This change in metabolism has the power to greatly reduce estrogen exposure as a risk for cancer.

This discovery proved that the metabolism and growth promoting activity of estrogen is modified by the intake of milligram amounts of dietary indoles from crucifers. When these cruciferous phytochemicals are added to the diet, estrogen action is regulated and its metabolism is shifted. This produces a predominance of 2-hydroxy and 2-methoxyestrogens. 4 These active metabolites have been called "good estrogens" 5, function as antioxidants 6, and have the power to eliminate damaged or cancerous cells throughout the body 7. Without these phytochemicals in the diet, there is increased production of a different, undesirable group of estrogen metabolites. These so-called "bad estrogens" act negatively to allow oxidation, to damage DNA, and to promote cancer 8.

A diet-derived imbalance in estrogen metabolism explains epidemiology showing a high prevalence of estrogen related disease, especially breast cancer, in societies consuming a diet low in total vegetable content.9 Supplemental use of diindolylmethane (DIM), the most active cruciferous indole, can restore and maintain a favorable balance of estrogen metabolites. Supplementation with DIM provides an innovative approach to reducing the estrogen-related risk of breast cancer. Therefore, DIM supplementation can increase the safety of estrogen replacement therapy in post menopausal women. In addition, aging-related alteration in estrogen metabolism is an under appreciated factor in men's health. DIM use by men promotes the same beneficial estrogen metabolism as seen in women. Improving estrogen balance in men may serve as a basis for enhancing prostate health.10

Diindolylmethane (DIM) and Estrogen Balance
Diindolylmethane (DIM) is the most active cruciferous substance for promoting beneficial estrogen metabolism in women and men.11 DIM is found in cruciferous vegetables including broccoli, cauliflower, cabbage and Brussels sprouts.12 DIM is formed from its precursor indole, Indole-3-carbinol (I3C), after the enzymatic release of I3C from parent glucosinolates found in all cruciferous vegetables. The supplemental use of DIM began with early experiments which demonstrated that animal diets with added DIM, like diets with added cruciferous vegetables 13, prevented chemically induced cancer.14 Pure DIM was first used in 1987 as a dietary supplement in animals, shown to be non-toxic, and to prevent breast cancer caused by the carcinogen, dimethylbenz(a)anthracene.15 Similarly, the initiation pathway to chemically induced colon cancer was inhibited with the DIM precursor, I3C.16 The mechanisms by which DIM prevents cancer in animals has subsequently been shown to involve a reduction in activity of the estrogen receptor system17, promotion of beneficial estrogen metabolism18, and support for selective apoptosis, or "programmed cell death" which removes damaged cells.19

Supplemental use of DIM in humans is effective in adjusting the pathways of estrogen metabolism to favor the production of 2-hydroxy estrogen metabolites.4 These shifts in estrogen metabolites were significant and showed an approximate 75% increase in production of 2-hydroxyestrone and a 50% decrease in 16-hydroxyestrone (See Figure 1.). An increased proportion of 2-hydroxy metabolites is correlated to protection from breast cancer. This relationship has been documented in several case-control studies. 20,21,22 Case-control studies have also documented that low levels of 2-hydroxy metabolites are associated with breast cancer in women 23, breast cancer in men 24, familial risk of breast cancer 25, uterine cancer 26, cervical cancer 27, and systemic lupus erythematosis 28. An increase in the rate of breast cancer has now been associated with lupus 29. Many established risk factors for breast cancer including obesity, high fat diets, and diets deficient in Omega 3 fatty acids have also been correlated with low 2-hydroxy estrone production.30 DIM is unique among all phytonutritionals with regard to its ability to favorably modify estrogen metabolism in the direction of greater 2-hydroxy estrogen production.

Extensive epidemiology has shown that consumption of crucifers in the human diet consistently reduces the occurrence of various cancers.31 With generous cruciferous intake the human diet provides up to 0.3 mg/kg of DIM. A recent human study was able to show that a daily intake of 500 grams of broccoli by volunteers slightly shifted the ratio of urinary estrogen metabolites, increasing urinary 2-hydroxyestrones in some of the subjects.32 Safe supplemental use of I3C was demonstrated in humans in 1992 33 , and confirmed in 199736. However, since I3C is highly unstable, its long term use in dietary supplements is of questionable value. I3C has been shown to be a precursor dietary indole, without activity until converted to DIM in the acid environment of the stomach. This process is inefficient, especially in the elderly, with diminished gastric acid production. Therefore, in addition to lacking shelf life, I3C has no biologic activity until converted to DIM. In contrast to I3C, DIM is highly stable, requires no conversion in the stomach, and is the most active cruciferous indole in promoting beneficial estrogen metabolism.34

Though stable, the extreme water insolubility of DIM in its pure crystalline form requires an absorption enhancing delivery system for use in dietary supplements. "IndolplexTM" is such a formulation containing microparticles of DIM associated with a soluble matrix which improves absorption. IndolplexTM demonstrates predictable and enhanced absorption of DIM in dietary supplements.35 IndolplexTM, containing standardized DIM, provides the phytochemical in a consistent, absorbable form which mimics the intake of DIM from the diet. These amounts of DIM relate to that found in large portions of Brussels sprouts or broccoli but exceed amounts conveniently reached from diet alone.35

Unlike soy isoflavones, genistein and daidzein, DIM is not an estrogen mimic or "phytoestrogen" and has no inherent estrogenic activity. DIM acts to balance the natural response to estrogen by adjusting the activity of metabolic cytochrome enzymes and specialized estrogen receptor molecules. In dividing cells this limits the growth promoting signal from estrogen by reducing the level of activity of the estrogen receptor system. In a complementary way, DIM also promotes the pathways of metabolism of estrogen within cells to favor the cancer preventive metabolites, 2-hydroxy and 2-methoxy estrogen.7,39 These metabolites further limit cell division and growth through influence on the cell cycle which determines growth and replication. 2-methoxy estrogen inhibits cell division by slowing the organization of tubulin, the subcellular cytoskeleton necessary for division of chromosomes.40 DIM's support for the protective mechanism of apoptosis, or programmed cell death, promotes beneficial elimination of damaged cells.41 The combination of these effects on cell behavior sets DIM apart from all other dietary substances and gives DIM a unique capability to promote beneficial actions of estrogen. Active apoptosis is central to preventing the initiation and promotion of breast, colon, and other cancers.42 Since these effects are specific to rapidly dividing cells, DIM does not prevent the beneficial effects of estrogen in supporting the health of the central nervous and skeletal systems. In essence, DIM creates a safer cellular environment for estrogen.

The Importance of DIM Supplementation in Pre and Postmenopausal Women
In premenopausal women, the first age-related hormonal imbalance involves a decreased production of progesterone. This reduction in progesterone output during the second half or luteal phase of the menstrual cycle, can cause irregular periods and contribute to premenstrual mood disorders. The "good estrogen" metabolites, 2-hydroxy and 2-methoxy estrogen are notable in that they stimulate increased progesterone production from ovarian cells 37,38. By promoting 2-hydroxy production, DIM supplementation can help support progesterone production and maintain progesterone levels through the peri-menopausal years. This balancing effect can benefit disorders associated with estrogen-progesterone imbalance including chronic, recurring breast pain, fibrocystic disease, and endometriosis.

In postmenopausal women, only about 20-30% of eligible women participate in long term use of supplemental estrogen. 43 This occurs despite compelling evidence as to the benefits of estrogen replacement, and relates to studies showing increased risk for breast and uterine cancer associated with prolonged HRT. 44 Even though this increase in risk is slight, the fearful nature of breast cancer makes hormonal replacement unacceptable to many women.45 Adding to this dilemma is the current wider availability of dehydroepiandrosterone (DHEA), a precursor adrenal steroid and the natural source of estrogen in post menopausal women.46 DHEA, sold as an over the counter dietary supplement in the USA, has been shown in long term clinical studies to promote bone mineralization without uterine stimulation47, and has demonstrated protection to breast tissue from cancer initiation and growth48. The importance of supplementation with DIM is that this approach can decrease estrogen-related breast cancer risk in the majority of women. This includes women taking estrogen, DHEA, phytoestrogen supplements, or no hormonal replacement at all.

DIM supplementation reduces undesirable metabolites of estrogen now known to be responsible for the cancer initiating and cancer promoting effects of estrogen.8 Supplemental DIM acts positively to support the pathways of estrogen metabolism which produce desirable metabolites associated with lower risk status for breast cancer and other estrogen-related disorders. The increased risk of breast cancer from postmenopausal estrogen administration can be eliminated by the complementary steps of adding cruciferous based supplements like DIM and reducing alcohol consumption to below 5 grams daily (equivalent to about 3 ounces of wine).49 The action of alcohol predictably raises circulating levels of estrogen from all sources by as much as 200% by interfering with its metabolism.50 If coupled with an unfavorable pathway of estrogen metabolism away from 2-hydroxy metabolites, alcohol can promote cancer whether or not supplemental estrogen is used. DIM supplementation is an effective and established means to assure a favorable pathway for estrogen metabolism. With its estrogen balancing effects, DIM provides a margin of safety and acts to reduce the negative consequences of estrogen elevation associated with moderate alcohol use.

Recent research clearly supports the benefits of maintaining estrogen levels in women as natural production declines with age. Post menopausal replacement of estrogen either through HRT or derived from DHEA is associated with a list of confirmed benefits.51 In addition to a decrease in overall mortality 52, estrogen replacement confers better memory and a lower risk of Alzheimer's dementia 53, stronger bones with fewer fractures54, and most importantly a 50% reduction in cardiovascular disease55, the number one threat to women's longevity. In addition, estrogen may be important in preventing osteoarthritis56, the most common cause of disability in women, and also may be important in reducing the occurrence of colon cancer57, the third most common cancer in women. There are also the benefits of more youthful skin, less vaginal dryness, increased libido, and less urinary incontinence. Following a long history of postmenopausal use in Europe, the addition of DHEA to regimens of hormonal supplementation is now advocated as one of the most advantageous source of estrogen for women.46
Use of phytonutritional supplements alone or in combination with hormonal replacement provides a new option for women which can minimize risks of breast and uterine cancer. This is based on strong evidence that DIM has cancer preventive activity.17 Dietary recommendations and applications of phytonutrition can now be combined to provide protection from estrogen risk while still taking advantage of estrogen's many benefits. Cruciferous intake has been shown to be protective against cancer in large population studies.31 These case-control studies support the importance of supplementation with specific dietary phytochemicals, like DIM.

The Importance of Healthy Estrogen Metabolism in Men's Health
In men, there is a new appreciation of the effects of changing estrogen metabolism with aging, now identified as "andropause". German researchers have clearly documented a dramatic, aging-related accumulation of estrogen in human prostate glands.58 This work correlated age, estrogen accumulation, and the presence of benign prostatic hypertrophy. This underscores the role of estrogen as a growth promoting hormone in men as well as women. Tissue accumulation of estrogen is a unique hallmark of andropause, distinct from estrogen deficiency which characterizes menopause. Recent work shows that estradiol, the active form of estrogen, provokes increases in prostate specific antigen (PSA) production in human prostate tissue.59 This increase in PSA is as great as that seen with testosterone. Increased PSA production was specifically inhibited by 2-methoxyestradiol, the beneficial estrogen metabolite whose production is promoted by DIM. 59

Accumulation of estrogen during andropause is amplified by obesity60 since fat tissue is the site of conversion of both testosterone and DHEA into estrogen. In case control studies, higher levels of circulating estrogen predict the degree of prostate enlargement. More importantly, increased estrogen levels have been repeatedly noted as a risk factor for early atherosclerosis and heart attack.61,62,63 The risks of elevated estrogen in men further correlate to decreased ability to dissolve blood clots.64 The specific deficiency in men of an active, beneficial metabolism of estrogen leading to 2-methoxy estrogens would explain many, if not all, of these observations.

Recent experimental work supports this connection between healthy estrogen metabolism and men's health. In studies culturing human vascular endothelial cells (HUVEC), it has been shown that 2-methoxy estradiol is a primary regulator of cell growth and apoptosis.65 Active and regulated apoptosis may contribute to the prevention of atherosclerotic plaque formation. At the basic level of lipoprotein status, 2-hydroxy and 2-methoxy estrogens are powerful antioxidants. In recent experiments, these metabolites, whose production is promoted by DIM, have been shown to prevent the oxidation of human lipoproteins.66 Lipoprotein oxidation is now accepted as an early, initiating event in atherosclerosis.
While it remains to be demonstrated through intervention studies that DIM supplementation can slow the progression of prostate disease and atherosclerosis, it is clear that DIM supplementation in men can beneficially shift estrogen metabolism. The supplemental use of DHEA has been advocated in men to replace dramatically decreased DHEA production with aging. A primary concern regarding this use of DHEA has been its partial conversion into estrogen. By promoting a healthy estrogen metabolism, use of DIM by men will increase the safety and benefits of long term supplementation with DHEA.

DIM and Environmental Estrogens
High fat diets and pesticide residues in food serve as additional sources of elevated estrogen exposure.75 This inadvertent estrogen exposure is increasing in both men and women throughout the world. In addition, high fat diets, especially those rich in animal fats or Omega 6 fatty acids can shift the metabolic pathways of estrogen towards 16-hydroxy metabolites. These "bad estrogens" are associated with higher rates of benign breast disease 67, and cancer in both women and men 24,26. A recent report of a large study of European women confirmed that dietary exposure to the estrogenic pesticide, Dieldren, was associated with increased risk of subsequent breast cancer.68 Exposure of breast cells in culture to organochlorine pesticides like Dieldrin increases the production of 16-hydroxy estrogen metabolites.69, 70 Alternatively, diets rich in Omega-3 fatty acids from fish 71 or supplemented with DIM produce more of the "good estrogens" identified as the 2-hydroxy metabolites of estradiol and estrone. Supplemental use of DIM promotes higher levels of 2-hydroxy estrogens. This use in animals has been shown to be associated with the prevention of spontaneous, estrogen related cancers of the breast and uterus 72,73 When tested in animals, DIM is unique in its effectiveness to favorably shift estrogen metabolism and decrease the activity of the estrogen receptor system.34 This activity of DIM relates to a blocking function toward the aryl hydrocarbon receptor which is then resistant to activation by pesticides.74 When made bioavailable with a delivery system such as IndolplexTM, DIM has been demonstrated to be effective in doses as low as 0.5 mg/kg/day of body weight as a means of increasing production of 2-hydroxy estrogen metabolites in men and women.35

Conclusion: Phytonutrition for Hormonal Support
Dietary supplementation with diindolylmethane (DIM) from cruciferous vegetables has established an important and effective nutritional approach to increasing the safety of estrogen. The availability of dietary supplements containing DIM provide an important new alternative in preventive nutrition and offer a source of support for the hormonal systems of men and women. To be effective, phytochemical supplements containing highly insoluble substances like DIM require absorption enhancing formulations. DIM supplementation can be combined with reduced alcohol intake to provide a dietary means of reducing the risk of breast and uterine cancer associated with HRT.49 The supplemental use of DIM allows women to promote and maintain a safer metabolism of estrogen. This expands the opportunities for women to derive the full preventive health benefits from long term hormonal replacement. DIM also increases the safety of exposure to estrogen derived from DHEA. This supports the rationale for long term supplementation with DHEA by both men and women. Documented, aging-related changes in men support their need for an improved metabolism of estrogen. 10 DIM use by men provides a promising dietary means to minimize the impact of increased estrogen on atherosclerosis and prostate disorders characteristic of andropause.58, These important benefits for successful aging in men and women all relate to an optimal and safer "estrogen balance".

Correspondence:
Michael A. Zeligs, M.D.
BioResponse
P.O. Box 288
Boulder, CO 80306

References
1. Adlercreutz H, Western diet and Western diseases: some hormonal and biochemical mechanisms and associations. Scand J Clin Lab Invest. 1990; 50 (Suppl 21): 3-23.

2. Michnovicz JJ, et al., Dietary and pharmacological control of estradiol metabolism in humans. Ann N Y Acad Sci. 1990; 595:291-9.

3. Michnovicz JJ, et al., Altered estrogen metabolism and excretion in humans following consumption of indole-3-carbinol. Nutr Cancer. 1991;16(1):59-66.

4.Michnovicz JJ, et al., Changes in levels of urinary estrogen metabolites after oral indole-3-carbinol treatment in humans. J Natl Cancer Inst. 1997 May 21; 89(10):718-23.

5. Bradlow HL, et al., 2-hydroxyestrone: the 'good' estrogen. J Endocrinol. 1996 Sep; 150 Suppl:S259-65.

6. Komura S, et al., Catecholestrogen as a natural antioxidant. Ann N Y Acad Sci. 1996 Jun 15; 786:419-429.

7. Zhu BT, et al., Is 2-Methoxyestradiol an endogenous estrogen metabolite that inhibits mammary carcinogenesis? Cancer Research 1998 June 1; 58:2269-2277

8. Service RF, New role for estrogen in cancer? Science 1998 March 13; 279:1631-1633.

9. Steinmetz KA, Vegetables, fruit, and cancer prevention: a review. J American Dietetic Assoc. 1996; 10: 1027-39.

10. Farnsworth WE, Roles of estrogen and SHBG in prostate physiology. The Prostate 1996; 28:17-23.

11. Zeligs MA, Diet and estrogen status: the cruciferous connection. J of Medicinal Food 1998 Nov 2; 1: 67-82.

12. Bradfield CA and Bjeldanes LF, High performance liquid chromatographic analysis of anticarcinogenic indoles in Brassica oleracea. J Agric. Food Chem. 1987; 35:46-49.

13. Boyd JN, et al, Modification by beet and cabbage diets of aflatoxin B1 induced rat plasma alpha-fetoprotein elevation, hepatic tumorigenesis, and mutagenicity of urine. Food Chen Toxicol. 1982; 20: 47.

14.Wattenberg LW and Loub WD, Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia by naturally occurring indoles. Cancer Research 1978; 38:1410-1413.

15. McDanell R, et al., Differential induction of mixed-function oxidase (MFO) activity in rat liver and intestine by diets containing processed cabbage: correlation with cabbage levels of glucosinolates and glucosinolate hydrolysis products. 1987 Food. Chem. Toxicicol.; 25: 363-368.

16. Guo D, Protection by chorophyllin and indole-3-carbinol against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced DNA adducts and colonic aberrant crypts in the F344 rat. Carcinogenesis 1995; 16: 2931-2937.

17. Chen I, et al., Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane. Carcinogenesis 1998; 19: 1631-1639.

18. Telang NT, et al., Inhibition of proliferation and modulation of estradiol metabolism: novel mechanisms for breast cancer prevention by the phytochemical indole-3-carbinol. Proceedings of the Society for Experimental Biology and Medicine 1997; 216: 246-252.

19. Gamet-Payrastre L, et al., Selective cytostatic and cytotoxic effects of glucosinolates hydrolysis products on human colon cancer cells in vitro. Anti-Cancer Drugs 1998; 9: 141-148.

20. Kabat GC, et al., Urinary estrogen metabolites and breast cancer: a case-control study.
Cancer Epidemiol Biomarkers Prev. 1997 Jul;6(7):505-9.

21. Meilahn EN, et al., Do urinary oestrogen metabolites predict breast cancer? Guernsey III cohort follow-up. British J of Cancer. 1998; 78: 1250-1255.

22. Ho GH, et al. Urinary 2/16 alpha-hydroxyestrone ratio: correlation with serum insulin-like growth factor binding protein-3 and a potential biomarker of breast cancer risk. Ann Acad Med Singapore 1998; 27:294-299.

23. Schneider J., et al., Abnormal oxidative metabolism of estradiol in women with breast cancer. Proc Natl Acad Sci USA 1982; 79: 3047-3051.

24. Zumoff B, et al., Estradiol transformation in men with breast cancer. J Clin Endocriol. Metab. 1966; 26: 960-966

25. Bradlow HL, et al., 16a hydroxylation of estradiol: a possible risk marker for breast cancer. Annals NY Acad. Sci. 1986; 464: 138-151.

26. Fishman, J, et al., Increased estrogen -16-hydroxylase activity in women with breast and endometrial cancer. Journal of Steroid Biochem. and Mol. Biol. 1984; 20: 1077-1081.

27. Sepkovic DW, et al., Estrogen metabolite ratios and risk assessment of hormone related cancers: assay validation and prediction of cervical cancer risk. Annals of the N.Y. Acad. of Science 1995; 768: 312-316.

28. Lahita, RG, et al., Increased 16a-hydroxylation of estradiol is systemic lupus erythematosus. J. Clin. Endocrinol. Metab. 1981; 53: 174-178.

29. Ramsey-Goldman R, et al., Increased risk of malignancy in patients with systemic lupus erythematosus. J. of Investigative Medicine 1998; 46:217-222.

30. Hershcopf RJ, et al., Obesity, diet, endogenous estrogens, and the risk of hormone-sensitive cancer. Amer. J. of Clinical Nutrition. 1987; 45 (Supplement 1): 283-289.

31. Verhoeven, DTH, et al., Epidemiological studies on brassica vegetables and cancer risk. Cancer Epidemiology, Biomarkers & Prevention. 1996; 5: 733-748.

32. Kall, M.A., et al. Effects of dietary broccoli on human drug metabolizing enzymes : evaluation of caffeine, oestrone and chlorzoxazone metabolism. Carcinogenesis 1996; 17: 793-799.

33. Bradlow HL, et al., Long-term responses of women to inole-3-carbinol or a high fiber diet. Cancer, Epidemiology, Biomarklers & Prevention 1994; 3:591-595.

34. Jellinck, PH, et al., Ah receptor binding properties of indole carbinols and induction of hepatic estradiol hydroxylation. Biochemical Pharmacology 1993; 45: 1129-1136.

35. Zeligs MA, Jacobs I, Facilitated absorption of a hydrophobic dietary supplement. Controlled Release Society Proceedings 1999: in press.

36. Wong YC, et al., Dose-ranging study of indole-3-carbinol for breast cancer prevention. J. of Cellular Biochemistry Supplenments. 1997; 28/29: 111-116.

37. Spicer LJ and Hammond JM, Comparative effects of androgens and catecholestrogens on progesterone production by porcine granulosa cells. Molecular and Cellular Endocrinology 1988; 56: 211-217.

38. Tekpetey FR,and Armstrong DT, Catecholestrogen modulation of steroid production by rat luteal cells: mechanism of action. Molecular and Cellular Endocrinology 1994; 101: 211-217.

39. Niwa T, et al., Alterations in estradiol metabolism in MCF-7 cells induced by treatment with indole-3-carbinol and related compounds.Steroids 1994 Sep; 59(9):523-7.

40. Klauber N, et al. Inhibition of angiogenesis and breast cancer in mice by the microtubule inhibitors 2-methoxyestradiol and taxol. Cancer Research 1997; 57:81-86.

41. Ge X, Yannai, S, Rennert, G, Gruener, N, and Fares, FA, 3,3'-Diindolylmethane induces apoptosis in human cancer cells. Biochemical and Biophysical Research Communications 1996; 228: 153-165.

42. Kerr JFR, et al., Apoptosis: its significance in cancer and cancer therapy. Cancer 1994; 73:3013-2026

43. Ettinger B, Overview of estrogen replacement therapy: a historical perspective. Proc. Soc. Exp. Biol. Med. 1998; 217:2-5.

44.Coldtiz GA, et al., The use of estrogens and progestins and the risk of breast cancer in postmenoapausal women. N.E.J.M. 1995; 332: 1589-93.

45. Colditz GA, Relationship between estrogen levels, use of hormone replacement therapy and breast cancer. J. Natl. Cancer Inst. 1998; 90: 814-23.

46. Labrie F., DHEA as physiological replacement therapy at menopause. J. Endocrinol. Invest. 1998; 21:399-401.

47.Labrie F, et al., Effect of 12-month dehydroepiandrosterone replacement therapy on bone, bagina, and endometrium in postmenopausal women. J. Clinical Endoc. Metab. 1997; 82:3498-3505.

48. Couillard S., et al., Inhibitory effect of dehydroepiandrosterone and of the novel antiestrogenEM-800 on the growth of hum ZR-75-1 breast cancer xenografts in nude mice. J. Natl. Cancer Inst. 1998;90:772-8.

49. Zumoff B, Does postmenopausal estrogen administration increase the risk of breast cancer? Contributions of animal, biochemical, and clinical investigative studies to a resolution of the controversey. Proc. Soc. Exp. Biol. Med. 1998; 217: 30-37.

50. Ginsburg EL, et al., Effects of alcohol ingestion on estrogens in postmenopausal women. JAMA 1996; 276:1747-51.

51. Calaf I, Alsina J, Benefits of hormone replacement therapy - overview and update. International J. of Fertility and Women's Medicine 1997; 42 Suppl 2: 329-46.

52. Grodstein F, et al., Postmenopausal hormone therapy and mortality. NEJM 1997; 336:1769-75

53. Inestrosa NC, et al., Cellular and molecular basis of estrogen's neuroprotection: potential relevance for Alzheimer's Disease. Molecular Neurobiology 1998; 17: 73-86.

54. Maxim P, et al., Fracture protection provided by long term estrogen replacement therapy. Osteoporosis Int. 1995; 5:23-29.

55. Manson JE, Postmenopausal hormone replacement and atherosclerotic disease. American Heart Journal 1994; 128:1337-43.

56. Spector TD, et al., Is hormone replacement therapy protective for hand and knee osteoarthritis in women? The Chingford study. Annals of the Rheumatic Diseases 1997; 56: 432-434.

57. Grodstein F, et al., Postmenopausal hormone use and risk for colorectal cancer and adenoma. Annals of Internal Medicine 1998; 128:705-12.

58.Krieg M., et al., Effect of aging on endogenous levels of 5-alpha-dihydrotestosterone, testosterone, estradiol and estrone in epithelium and stroma of normal and hyperplastic human prostate. J. Clin. Endocrinol. Metab 1993; 77: 375-381

59. Nakhla AM, et al., Estradiol activates the prostate androgen receptor and prostate-specific antigen secretion through the intermediacy of sex hormone binding globulin. J. of Biological Chemistry 1997; 272: 6838-6841

60. Brind J, Obese men have elevated plasma levels of estrone sulfate. Int. J. of Obesity 1990; 14: 483-486.

61. Klaiber EL, et al., Serum estrogen levels in men with acute myocardial infarction. American Journal of Medicine 1982; 73: 872-881.

62. Philips GB, Evidence for hyperoestrogenaemia as a risk factor for myocardial infarction in men. Lancet 1976; 2(7975): 14-18.

63. Phillips GB, et al., Association of hyperestrogenemia and coronary heart disease in men in the Framingham cohort. American J. of Medicine 1983; 74: 863-869.

64. Andersen P, et al., Reduced fibrinolytic capacity associated with low ratio of serum testosterone to oestradiol in healthy coronary high risk men. Scand. J. Haematol. 1983; 30 (Suppl 39): 53-57.

65. Reiser F., et al., Inhibition of normal and experimental angiotumor endothelial cell proliferation and cell cycle progression by 2-methoxyestradiol. Proc. Soc. Exp. Biol. Med. 1998; 219(3): 211-16.

66. Seeger H., et al., The effect of estradiol metabolites on the susceptibility of low density lipoprotein to oxidation. Life Sciences 1997; 61:865-868

67. Rose DP, et al., Effect of a low fat diet on hormone levels in women with cystic breast disease. II Serum radioimmunoassayable prolactin and growth hormone and bioactive lactogenic hormones. J. Natl. Cancer Inst. 1987; 78:627-631.

68. Hoyer AP, et al., Organochlorine exposure and risk of breast cancer. Lancet 1998 Dec 5; 352:1816-1820.

69. Bradlow HL, et al., Effects of pesticides on the ratio of 16 alpha/2-hydroxyestrone: a biologic marker of breasst cancer risk.Environ Health Perspect. 1995 Oct;103 Suppl 7:147-50.

70. Bradlow HL, et al. Role of the estrogen receptor in the action of organochlorine pesticides on estrogen metabolism in human breast cancer cell lines. Sci Total Environ. 1997 Dec 3; 208(1-2): 9-14.

71. Osborne MP, et al., Omega-3-fatty acids: modulation of estrogen metabolism and potential for breast cancer prevention. Cancer Investigation 1988; 8:629-631.

72. Bradlow HL, Michnovicz JJ, Telang NT, and Osborne MP, Effects of dietary indole-3-carbinol on estradiol metabolism and spontaneous mammary tumors in mice. Carcinogenesis 12, 1571-1574

73. Kojima T., et al., Chemoprevention of spontaneous endometrial cancer in female donryu rats by dietary indole-3-carbinol. Cancer Research 1994; 54: 1446-1449.

74. Chen I., et al., Indole-3-carbinol and diindolylmethane as aryl hydrocarbon (AH) receptor agonists and antagonists in T47D human breast cancer cells. Biochemical Pharmacology 1996; 51:1069-1076.

75. Davis DL, Bradlow HL, Can environmental estrogens cause breast cancer? Sci Am. 1995 Oct. 273(4): 167-72. Review.