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Micronutrient Research for Optimum Health

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Indole-3-Carbinol
Summary Indole-3-carbinol (I3C) is derived from the hydrolysis (breakdown) of glucobrassicin, a compound found in cruciferous vegetables. (More Information) In the acidic environment of the stomach, I3C molecules can combine with each other to form a number of biologically active acid condensation products, such as 3,3'diindolylmethane (DIM). (More Information) I3C has been found to inhibit the development of cancer in animals when given before or at the same time as a carcinogen. However, in some cases, I3C enhanced the development of cancer in animals when administered after a carcinogen. (More Information) The contradictory results of animal studies have led some experts to caution against the widespread use of I3C and DIM supplements for cancer prevention in humans until their potential risks and benefits are better understood. (More Information) Although I3C and DIM supplementation have been found to alter urinary estrogen metabolite profiles in women, the effects of I3C and DIM on breast cancer risk are not known. (More Information) Results of small preliminary trials in humans suggest that I3C supplementation may help treat conditions related to human papilloma virus (HPV) infection, such as cervical intraepithelial neoplasia (CIN) and recurrent respiratory papillomatosis (RRP). However, randomized controlled trials are needed to determine whether I3C supplementation is beneficial. (More Information) Introduction Cruciferous vegetables differ from other classes of vegetables in that they are rich sources of sulfur -containing compounds known as glucosinolates (see Cruciferous Vegetables). Because epidemiological studies provide some evidence that diets rich in cruciferous vegetables are associated with lower risk of several types of cancer, scientists are interested in the potential cancer-preventive activities of compounds derived from glucosinolates (1). Among these compounds is indole-3-carbinol (I3C), a compound derived from the enzymatic hydrolysis (breakdown) of an indole glucosinolate, commonly known as glucobrassicin (2). Metabolism and Bioavailability A number of commonly consumed cruciferous vegetables, including broccoli, Brussels sprouts, and cabbage, are good sources of glucobrassicinthe glucosinolate precursor of I3C. Myrosinase, an enzyme that catalyzes the hydrolysis of glucosinolates, is physically separated from glucosinolates

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in intact plant cells (3). When plant cells are damaged, as when cruciferous vegetables are chopped or chewed, the interaction of myrosinase and glucobrassicin results in the formation of I3C (figure 1). In the acidic environment of the stomach, I3C molecules can combine with each other to form a complex mixture of biologically active compounds, known collectively as acid condensation products (4). Although numerous acid condensation products of I3C have been identified, some of the most prominent include the dimer 3,3'-diindolylmethane (DIM) and a cyclic trimer (CT) (figure 2). The biological activities of individual acid condensation products differ from those of I3C and are responsible for the biological effects attributed to I3C (5). When plant myrosinase is inactivated (e.g., by boiling), glucosinolate hydrolysis still occurs to a lesser degree, due to the myrosinase activity of human intestinal bacteria (6). Thus, when cruciferous vegetables are cooked in a manner that inactivates myrosinase, glucobrassicin hydrolysis by intestinal bacteria still results in some I3C formation (see Food Sources). However, acid condensation products are less likely to form in the more alkaline environment of the intestine. Biological Activities Effects on Biotransformation Enzymes Involved in Carcinogen Metabolism Biotransformation enzymes play major roles in the metabolism and elimination of many biologically active compounds, including steroid hormones, carcinogens, toxins, and drugs. In general, phase I biotransformation enzymes, including the cytochrome P450 (CYP) family, catalyze reactions that increase the reactivity of hydrophobic (fat-soluble) compounds, which prepares them for reactions catalyzed by phase II biotransformation enzymes. Reactions catalyzed by phase II enzymes generally increase water solubility and promote the elimination of these compounds (7). Acid condensation products of I3C, particularly DIM and indole[3,2-b]carbazole (ICZ), can bind to a protein in the cytoplasm of cells called the aryl hydrocarbon receptor (AhR) (5, 8). Binding allows the AhR to enter the nucleus where it forms a complex with the Ahr nuclear translocator (Arnt) protein. This Ahr/Arnt complex binds to specific DNA sequences in genes known as xenobiotic response elements (XRE) and enhances their transcription (9). Genes for a number of CYP enzymes and several phase II enzymes are known to contain XREs. Thus, oral consumption of I3C results in the formation of acid condensation products that can increase the activity of certain phase I and phase II enzymes (8, 10, 11). Increasing the activity of biotransformation enzymes is generally considered a beneficial effect because the elimination of potential carcinogens or toxins is enhanced. However, there is a potential for adverse effects because some procarcinogens require biotransformation by phase I enzymes to become active carcinogens (12). Alterations in Estrogen Activity and Metabolism Endogenous estrogens, including 17beta-estradiol, exert their estrogenic effects by binding to estrogen receptors (ERs). Within the nucleus, the estrogen-ER complex can bind to DNA sequences in genes known as estrogen response elements (EREs), recruit coactivator molecules, and thus enhance the transcription of estrogen-responsive genes (13). Some ER-mediated effects, such as those that promote cellular proliferation in the breast and uterus, can increase the risk of developing estrogen-sensitive cancers (14). Effects on Estrogen Receptor Activity When added to breast cancer cells in culture, I3C has been found to inhibit the transcription of estrogen-responsive genes stimulated by 17beta-estradiol (15, 16). Acid condensation products of I3C that bind and activate AhR may also inhibit the transcription of estrogen-responsive genes by competing for coactivators or increasing ER degradation (9, 17). In contrast, some studies in cell culture (18, 19) and animal models (20) have found that acid condensation products of I3C actually enhance the transcription of estrogen-responsive genes. Further research is needed to determine the nature of the stimulatory and inhibitory effects of I3C and its acid condensation

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products on estrogen-responsive gene transcription under conditions that are relevant to human cancer risk (see Cancer below). Effects on Estrogen Metabolism The endogenous estrogen 17beta-estradiol can be irreversibly metabolized to 16alphahydroxyestrone (16OHE1) or 2-hydroxyestrone (2OHE1). In contrast to 2OHE1, 16OHE1 is highly estrogenic and has been found to stimulate the proliferation of several estrogen-sensitive cancer cell lines (21, 22). It has been hypothesized that shifting the metabolism of 17beta-estradiol toward 2OHE1, and away from 16OHE1, could decrease the risk of estrogen-sensitive cancers, such as breast cancer (23). In controlled clinical trials, oral supplementation with 300-400 mg/day of I3C has consistently increased urinary 2OHE1 levels or urinary 2OHE1:16OHE1 ratios in women (24 -29). Supplementation with 108 mg/day of DIM also increased urinary 2OHE1 levels in postmenopausal women (30). However, the relationship between urinary 2OHE1:16OHE1 ratios and breast cancer risk is not clear. Although women with breast cancer had lower urinary ratios of 2OHE1:16OHE1 in several small case-control studies (31-33), larger case-control and prospective cohort studies have not found significant associations between urinary 2OHE1:16OHE1 ratios and breast cancer risk (34-36). Induction of Cell Cycle Arrest Once a cell divides, it passes through a sequence of stagescollectively known as the cell cycle before it divides again. Following DNA damage, the cell cycle can be transiently arrested at damage checkpoints, which allows for DNA repair or activation of pathways leading to cell death (apoptosis) if the damage is irreparable (37). Defective cell cycle regulation may result in the propagation of mutations that contribute to the development of cancer. The addition of I3C to prostate and breast cancer cells in culture has been found to induce cell cycle arrest (38, 39). However, the physiological relevance of these cell culture studies is unclear since little or no I3C is available to tissues after oral administration (see Metabolism and Bioavailability) (40). Induction of Apoptosis Unlike normal cells, cancerous cells lose their ability to respond to death signals that initiate apoptosis. I3C and DIM have been found to induce apoptosis when added to cultured prostate (38), breast (41- 43), pancreatic (44), and cervical cancer cells (45). Inhibition of Tumor Invasion and Angiogenesis Limited evidence in cell culture experiments suggests that I3C and DIM can inhibit the invasion of normal tissue by cancer cells (46) and also inhibit the development of new blood vessels (angiogenesis) required by rapidly growing tumors (47, 48). Disease Prevention Cancer Epidemiological Studies Epidemiological studies provide some support for the hypothesis that higher intakes of cruciferous vegetables are associated with lower risk for some types of cancer (49). However, cruciferous vegetables are relatively good sources of other phytonutrients that may have protective effects against cancer, including vitamin C, folate, selenium, carotenoids, and fiber (see Cruciferous Vegetables). Moreover, cruciferous vegetables provide a variety of glucosinolates, in addition to indole-3-carbinol, that may be hydrolyzed to a variety of potentially protective isothiocyanates (e.g., sulforaphane; see Isothiocyanates) (50). Consequently, evidence for an inverse association

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between cruciferous vegetable intake and cancer risk provides relatively little information about the specific effects of indole-3-carbinol on cancer risk. Animal Studies In most animal models, exposure to a chemical carcinogen is required to cause cancer. When administered before or at the same time as the carcinogen, oral I3C has been found to inhibit the development of cancer in a variety of animal models and tissues, including cancers of the mammary gland (breast) (51, 52), uterus (53), stomach (54), colon (55, 56), lung (57), and liver (58, 59). However, a number of studies have found that I3C actually promoted or enhanced the development of cancer when administered chronically after the carcinogen (post-initiation). The cancer-promoting effects of I3C were first reported in a trout model of liver cancer (60, 61). However, I3C has also been found to promote cancer of the liver (62-64), thyroid (64), colon (65, 66), and uterus (67) in rats. More recently, inclusion of I3C in the maternal diet was found to protect the offspring from lymphoma and lung tumors induced by dibenzo[a,l]pyrene, a polycyclic aromatic hydrocarbon (68). Polycyclic aromatic hydrocarbons are chemical pollutants formed during incomplete combustion of organic substances, such as coal, oil, wood, and tobacco (69). Although the long-term effects of I3C supplementation on cancer risk in humans are not known, the contradictory results of animal studies have led several experts to caution against the widespread use of I3C and DIM supplements in humans until their potential risks and benefits are better understood (62, 70, 71). Disease Treatment Diseases Related to Human Papilloma Virus Infection Cervical Intraepithelial Neoplasia Infection with certain strains of human papilloma virus (HPV) is an important risk factor for cervical cancer (72). Transgenic mice that express cancer-promoting HPV genes develop cervical cancer with chronic 17beta-estradiol administration. In this model, feeding I3C markedly reduced the number of mice that developed cervical cancer (73). A small placebo-controlled trial in women examined the effect of oral I3C supplementation on the progression of precancerous cervical lesions classified as cervical intraepithelial neoplasia (CIN) 2 or CIN 3 (74). After 12 weeks, four out of the eight women who took 200 mg/day had complete regression of CIN and four out of the nine who took 400 mg/day had complete regression, while none of the ten who took a placebo had complete regression. HPV was present in seven out of the ten women in the placebo group, seven out of eight women in the 200 mg I3C group, and eight out of nine women in the 400 mg I3C group (74). Although these preliminary results are encouraging, larger controlled clinical trials are needed to determine the efficacy of I3C supplementation for preventing the progression of precancerous lesions of the cervix (75). Vulvar Intraepithelial Neoplasia HPV infection can also lead to vulvar intraepithelial neoplasia (VIN) (76). A small randomized trial in 12 women with VIN found that supplementation with 200 mg/day or 400 mg/day of I3C for six months improved overall symptoms and decreased lesion size and degree of aggressive histopathology (77). While the results of this preliminary trial are promising, more clinical trials are needed to determine whether I3C might be an effective treatment for VIN. Recurrent Respiratory Papillomatosis Recurrent respiratory papillomatosis (RRP) is a rare disease of children and adults, which are characterized by generally benign growths (papillomas) in the respiratory tract caused by HPV infection (78). These papillomas occur most commonly on or around the vocal cords in the larynx

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(voice box), but they may also affect the trachea, bronchi, and lungs. The most common treatment for RRP is surgical removal of the papillomas. Since papillomas often recur, adjunct treatments may be used to help prevent or reduce recurrences (79). In immune-compromised mice transplanted with HPV-infected laryngeal tissue, only 25% of the mice fed I3C developed laryngeal papillomas compared to 100% of the control mice (80). In a small observational study of RRP patients, increased ratios of urinary 2OHE1:16OHE1 ratios resulting from increased cruciferous vegetable consumption were associated with less severe RRP (81). Most recently, an uncontrolled pilot study examined the effect of I3C supplementation (400 mg/day for adults and 10 mg/kg daily for children) on papilloma recurrence in RRP patients (82). Over a 5-year follow-up period, 11 of the original 49 patients experienced no recurrence, ten experienced a reduction in the rate of recurrence, 12 experienced no improvement, and 12 were lost to follow-up (83). Although the low toxicity of I3C makes it an attractive adjunct therapy for RRP, controlled clinical trials are needed to determine whether I3C is effective in preventing or reducing the recurrence of respiratory papillomas. Systemic Lupus Erythematosus Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by chronic inflammation that may result in damage to the joints, skin, kidneys, heart, lungs, blood vessels, or brain (84). Estrogen is thought to play a role in the pathology of SLE because the disorder is much more common in women than men, and its onset is most common during the reproductive years when endogenous estrogen levels are highest (85). The potential for I3C supplementation to shift endogenous estrogen metabolism toward the less estrogenic metabolite 2OHE1, and away from the highly estrogenic metabolite 16OHE1 (see Estrogen Metabolism), led to interest in its use in SLE (24). In an animal model of SLE, I3C feeding decreased the severity of renal (kidney) disease and prolonged survival (86). A small uncontrolled trial of I3C supplementation (375 mg/day) in female SLE patients found that I3C increased urinary 2OHE1:16OHE1 ratios, but the trial found no significant change in SLE symptoms after three months (86). Controlled clinical trials are needed to determine whether I3C supplementation will have beneficial effects in SLE patients. Sources Food Sources Glucobrassicin, the glucosinolate precursor of I3C, is found in a number of cruciferous vegetables, including broccoli, Brussels sprouts, cabbage, cauliflower, collard greens, kale, kohlrabi, mustard greens, radish, rutabaga, and turnip (87, 88). Although glucosinolates are present in relatively high concentrations in cruciferous vegetables, glucobrassicin makes up only about 8-12% of the total glucosinolates (89). Total glucosinolate contents of selected cruciferous vegetables are presented in the table below (90). The amount of indole-3-carbinol formed from glucobrassicin in foods is variable and depends, in part, on the processing and preparation of foods. Effects of Cooking Glucosinolates are water-soluble compounds that may be leached into cooking water. Boiling cruciferous vegetables from 9-15 minutes resulted in 18-59% decreases in the total glucosinolate content of cruciferous vegetables (90). Cooking methods that use less water, such as steaming or microwaving, may reduce glucosinolate losses. Some cooking practices, including boiling (91), steaming (92), and microwaving at high power (850-900 watts) (93, 94), may inactivate myrosinase, the enzyme that catalyzes glucosinolate hydrolysis. Even in the absence of plant myrosinase activity, the myrosinase activity of human intestinal bacteria results in some glucosinolate hydrolysis (6). However, studies in humans have found that inactivation of myrosinase in cruciferous vegetables substantially decreases the bioavailability of glucosinolate hydrolysis products known as isothiocyanates (91-93). Since the formation of I3C also depends on glucosinolate hydrolysis, it is very likely that the bioavailability of I3C and its acid condensation products would also be decreased by myrosinase inactivation.

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Glucosinolate Content of Selected Cruciferous Vegetables (90) Food (raw) Garden cress Mustard greens Brussels sprouts Horseradish Kale Watercress Turnip Cabbage, savoy Cabbage, red Broccoli Kohlrabi Bok choi (pak choi) Cauliflower Serving cup (25 g) cup, chopped (28 g) cup (44 g) 1 tablespoon (15 g) 1 cup, chopped (67 g) 1 cup, chopped (34 g) cup, cubes (65 g) cup, chopped (45 g) cup, chopped (45 g) cup, chopped (44 g) cup, chopped (67 g) cup, chopped (35 g) cup, chopped (50 g) Total Glucosinolates (mg/g of food) 3.9 2.8 2.4 1.6 1.0 0.9 0.9 0.8 0.6 0.6 0.5 0.5 0.4

Supplements Indole-3-Carbinol (I3C) I3C is available without a prescription as a dietary supplement. I3C supplementation increased urinary 2OHE1 levels in adults at doses of 300-400 mg/day (28). I3C doses of 200 mg/day or 400 mg/day improved the regression of cervical intraepithelial neoplasia (CIN) in a preliminary clinical trial (74). I3C in doses up to 400 mg/day has been used to treat recurrent respiratory papillomatosis (82, 83). These supplemental levels are well above levels dietary levels, which commonly range from 20-120 mg daily (95). 3,3'-Diindolylmethane (DIM) DIM is available without a prescription as a dietary supplement. In a small clinical trial, DIM supplementation at a dose of 108 mg/day for 30 days increased urinary 2OHE1 excretion in postmenopausal women with a history of breast cancer (30). Safety Adverse Effects Slight increases in the serum concentrations of a liver enzyme (alanine aminotransferase; ALT) were observed in two women who took unspecified doses of I3C supplements for four weeks (28). One person reported a skin rash while taking 375 mg/day of I3C (24). High doses of I3C (800 mg/day) were associated with symptoms of disequilibrium and tremor, which resolved when the dose was decreased (82). I3C supplementation enhanced the development of cancer in some animal models when given after the carcinogen (62, 64, 65, 67) (see Cancer). The effects of I3C or DIM supplementation on cancer risk in humans are not known. Pregnancy and Lactation The safety of I3C or DIM supplements during pregnancy or lactation has not been established.

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Drug Interactions No drug interactions in humans have been reported. However, preliminary evidence that I3C and DIM can increase the activity of CYP1A2 (96, 97) suggests the potential for I3C or DIM supplementation to decrease serum concentrations of medications metabolized by CYP1A2. Both I3C and DIM modestly increase the activity of CYP3A4 in rats when administered chronically (98). This observation raises the potential for adverse drug interactions in humans since CYP3A4 is involved in the metabolism of approximately 50% of therapeutic drugs. References Written in July 2005 by: Jane Higdon, Ph.D. Linus Pauling Institute Oregon State University Updated in December 2008 by: Victoria J. Drake, Ph.D. Linus Pauling Institute Oregon State University Reviewed in December 2008 by: David E. Williams, Ph.D. Principal Investigator, Linus Pauling Institute Professor, Department of Environmental and Molecular Toxicology Oregon State University Copyright 2005-2012 Linus Pauling Institute Disclaimer The Linus Pauling Institute Micronutrient Information Center provides scientific information on health aspects of micronutrients and phytochemicals for the general public. The information is made available with the understanding that the author and publisher are not providing medical, psychological, or nutritional counseling services on this site. The information should not be used in place of a consultation with a competent health care or nutrition professional. The information on micronutrients and phytochemicals contained on this Web site does not cover all possible uses, actions, precautions, side effects, and interactions. It is not intended as medical advice for individual problems. Liability for individual actions or omissions based upon the contents of this site is expressly disclaimed.

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