Induction of estradiol metabolism by dietary indole-3-carbinol in humans.

Dietary indoles in cruciferous vegetables induce cytochrome P450 enzymes and have prevented tumors in various animal models. Because estradiol metabolism is also cytochrome P450 mediated and linked to breast cancer risk, indoles may similarly reduce estrogen-responsive tumors in humans. We examined several indoles in female Sprague-Dawley rats for induction of hepatic estradiol 2-hydroxylation. The most potent inducer, indole-3-carbinol, was administered to humans (500 mg daily for 1 wk). It significantly increased the extent (mean +/- SEM) of estradiol 2-hydroxylation from 29.3% +/- 2.1% to 45.6% +/- 2.1% (P less than .001). These results indicate that indole-3-carbinol strongly influences estradiol metabolism in humans and may provide a new chemopreventive approach to estrogen-dependent diseases.

Changes in levels of urinary estrogen metabolites after oral indole-3-carbinol treatment in humans.

BACKGROUND: The oxidative metabolism of estrogens in humans is mediated primarily by cytochrome P450, many isoenzymes of which are inducible by dietary and pharmacologic agents. One major pathway, 2-hydroxylation, is induced by dietary indole-3-carbinol (I3C), which is present in cruciferous vegetables (e.g., cabbage and broccoli). PURPOSE: Because the pool of available estrogen substrates for all pathways is limited, we hypothesized that increased 2-hydroxylation of estrogens would lead to decreased activity in competing metabolic pathways. METHODS: Urine samples were collected from subjects before and after oral ingestion of I3C (6-7 mg/kg per day). In the first study, seven men received I3C for 1 week; in the second study, 10 women received I3C for 2 months. A profile of 13 estrogens was measured in each sample by gas chromatography-mass spectrometry. RESULTS: In both men and women, I3C significantly increased the urinary excretion of C-2 estrogens. The urinary concentrations of nearly all other estrogen metabolites, including levels of estradiol, estrone, estriol, and 16alpha-hydroxyestrone, were lower after I3C treatment. CONCLUSIONS: These findings support the hypothesis that I3C-induced estrogen 2-hydroxylation results in decreased concentrations of several metabolites known to activate the estrogen receptor. This effect may lower estrogenic stimulation in women. IMPLICATIONS: I3C may have chemopreventive activity against breast cancer in humans, although the long-term effects of higher catechol estrogen levels in women require further investigation.

19-Hydroxylation and aromatization of androgens in the developing rat brain.

Androgen aromatization in the human placenta proceeds through two successive hydroxylations at C-19, the products of which are then virtually completely converted to estrogens. In the neonatal rat brain, however, 19-hydroxylation has been shown to exceed significantly subsequent aromatization, suggesting that formation of 19-hydroxylated androgen metabolites might be important in brain differentiation in this species. Using [19-3H3]androstenedione, we found that the surplus activity of 19-hydroxylase relative to aromatase was independent of age, sex, and androgen substrate concentration, despite 100-fold differences in tissue aromatase activity during the course of development. In addition, the surplus 19-hydroxylation was not affected by several agents which otherwise decreased or increased the activity of the aromatase enzyme, including metyrapone, KCN, and cytochrome P-450 reductase, the latter indicating that the failure of the 19-hydroxylated products to proceed to aromatization was not due to a deficit of reducing equivalents. 19-Hydroxylation of androgens in the rat brain is a quantitatively significant metabolic pathway in this tissue, although present data do not confirm the existence of a steroid C-19 hydroxylase in the brain separate from that involved in aromatization.

Long-term responses of women to indole-3-carbinol or a high fiber diet.

We test the hypothesis that the estrogen metabolite ratio 2-OH-estrone:estriol can be raised via dietary indole-3-carbinol (I3C) and that this higher ratio can be sustained over a 3-month test period. We also explore the possible role of pure fiber on estradiol metabolism. Using a randomized clinical trial with three arms, each containing 20 subjects, arm 1 received 400 mg/day of I3C daily for 3 months, arm 2 received 20 g of alpha-cellulose daily for the same time period as a source of added fiber, and arm 3 received a placebo dose. Blood levels of a variety of biochemical parameters were measured. The urinary 2-OH-estrone:estriol estrogen metabolite ratio was measured monthly at the same time of the menstrual cycle. While no changes were observed in the control and alpha-cellulose-treated arms, a substantial mean increase in the ratio was observed in the I3C-treated arm at month 1; that increase was maintained over the 3-month time period. Three of the 20 subjects in this I3C-treated group differed from the others in that no significant change in the metabolite ratio was observed at any time point. The results suggest that I3C can serve to increase the 2-OH-estrone:estriol metabolite ratio in a sustained manner without detectable side effects and that some individuals may be resistant to such change.

Oxidative metabolism of estrogens in rat intestinal mitochondria.

Intestinal epithelial cells are capable of metabolizing a wide variety of exogenous substrates. To determine how this metabolic capacity may affect endogenous substances such as steroid hormones, we examined the ability of rat gut epithelial preparations to hydroxylate estradiol at the C-2 position. Utilizing a site-specific tritium exchange assay, an active estrogen 2-hydroxylase system was shown to be localized to gut mitochondria throughout the intestine, with enzymatic activities comparable to the activity in crude hepatic homogenates of non-induced animals (0.2 nmol/min/mg protein). Gas chromatography-mass spectrometry confirmed the formation of C-2 hydroxylated estrogens by these mitochondrial preparations. The enzyme system was shown to involve a saturable monooxygenase, utilizing NADH (preferably) or NADPH in a protein- and time-dependent fashion. The Michaelis-Menten constant for this pathway was approximately 150 microM. Enzyme activity decreased by 20% in the presence of carbon monoxide, and was largely unaffected by organic P450 inhibitors such as alpha-naphthoflavone, metyrapone, and SKF-525A. These studies suggest that intestinal mitochondria are able to contribute to the oxidative metabolism of endogenous estrogens circulating within the enterohepatic pool.

Ah receptor binding properties of indole carbinols and induction of hepatic estradiol hydroxylation.

The effect of route of administration on the ability of indole-3-carbinol (13C), an anticarcinogen present in cruciferous vegetables, to induce estradiol 2-hydroxylase (EH) in female rat liver microsomes was investigated and compared to that of its main gastric conversion product, 3,3'-diindolylmethane (DIM). This dimer was more potent than 13C after either oral or intraperitoneal administration and was also a better in vitro inhibitor of EH in control and 13C-induced hepatic microsomes. The induction of both CYP1A1 and 1A2 in about equal amounts by 13C and DIM as well as of CYP2B1/2 was demonstrated using monoclonal antibodies. DIM, isosafrole, beta-naphthoflavone, 3-methylcholanthrene and naringenin added in vitro inhibited EH strongly in induced microsomes but gestodene was a better inhibitor of estrogen 2-hydroxylation in liver microsomes from untreated female rats. The binding affinities of 13C and DIM to the Ah receptor were compared to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by competition studies, and the IC50 values were shown to be 2.0 x 10(-9) M, 5.0 x 10(-5) M and 2.3 x 10(-3) M for TCDD, DIM and 13C, respectively. The ability of 13C or DIM to cause in vitro transformation of the Ah receptor to a form able to bind to the dioxin-responsive element-3 (DRE3) was compared to that of TCDD and shown to parallel their abilities to compete for binding of [3H]TCDD to the Ah receptor. These experiments confirm and extend the proposals that dietary indoles induce specific cytochrome P450s in rat liver by a mechanism possibly involving the Ah receptor. The induced monooxygenases, in turn, increase the synthesis of 2-hydroxylated estrogens in the competing pathways of 2- and 16 alpha-hydroxylation which decreases the levels of 16 alpha-hydroxyestrone able to form stable covalent adducts with proteins including the estrogen receptor. Such steroid-protein interaction has been correlated with mammary carcinogenesis.

Dietary and pharmacological control of estradiol metabolism in humans.

Clinical research has demonstrated that increased or decreased estradiol 2-hydroxylation can easily be achieved with a number of experimental approaches. In contrast, estradiol 16 alpha-hydroxylation, which may have potentially deleterious effects in estrogen-dependent tissues, cannot be readily altered. Predictable hormonal consequences have thus far been found in response to the modification of 2-hydroxylation. This approach offers promise as a method for specifically altering the risk for diseases associated with either too little estrogen (osteoporosis) or too much estrogen (breast and uterine cancer).

Cimetidine inhibits catechol estrogen metabolism in women.

Chronic cimetidine use in men is associated with hyperestrogenic side effects such as gynecomastia, which may be linked to inhibition of estradiol 2-hydroxylation. As this property of the drug might be helpful in hypoestrogenic states such as osteoporosis, we investigated the effect of cimetidine on estradiol metabolism in premenopausal and postmenopausal women. Using an in vivo radiometric assay, we found that the extent of estradiol 2-hydroxylation in premenopausal women (n = 9) was decreased by a 1-month course of cimetidine, 800 mg twice daily (44.0% +/- 3.5% v 31.2% +/- 4.1%, P less than .001). Among premenopausal smokers (n = 3), the response to cimetidine was approximately the same as nonsmokers. Serum estradiol levels (follicular phase) in these women were unaltered by cimetidine after 1 month, while concentrations of sex hormone-binding globulin (SHBG) were decreased by 30% (P = .018). Postmenopausal women (n = 5) initially received a lower dose of cimetidine (600 mg twice daily) for 2 weeks, followed by a higher dose (1200 mg twice daily) for another 2 weeks. The extent of estradiol 2-hydroxylation was significantly reduced by the low dose (44.4% +/- 4.5% v 24.3% +/- 3.0%, P less than .005), with minimal further reduction after the high dose (21.7% +/- 1.6%). After 4 weeks of cimetidine treatment, serum estradiol levels increased significantly from 30.0 +/- 6.4 to 59.8 +/- 13.1 pg/mL (P = .033), while SHBG was unaffected. Cimetidine was found to have little effect on selected biochemical indices of bone and calcium metabolism in both groups of women.(ABSTRACT TRUNCATED AT 250 WORDS)

Cigarette smoking alters hepatic estrogen metabolism in men: implications for atherosclerosis.

Studies of steroids and plasma lipoproteins in male cigarette smokers reveal that smoking is associated with an increase in peripheral estrogens and a decrease in high-density lipoprotein-cholesterol (HDL-C). We hypothesized that the lower HDL-C in this setting results in part from induction of the hepatic metabolic pathway that inactivates estrogen. This pathway, estradiol 2-hydroxylation, produces the peripherally inactive catechol estrogens 2-hydroxyesterone and 2-methoxyestrone. We used an in vivo radiometric method to assess 2-hydroxylation in 20 male smokers and 16 nonsmokers. The extent of the reaction (+/- SEM) was significantly higher among the smokers (43.3% +/- 1.9% v 24.6% +/- 1.9%, P less than .001). Smokers also excreted more urinary 2-hydroxyestrone (10.4 +/- 1.3 micrograms/g creatinine v 6.3 +/- 0.73 micrograms/g in nonsmokers, P = .011). The ratio of urinary 2-hydroxyestrone to estriol was higher on average among smokers (1.46 +/- 0.19 v 0.81 +/- 0.11, P = .006), and individual values correlated well with the radiometric test (r = .71, P less than .002). These data indicate that smoking is associated with significantly increased estrogen 2-hydroxylation in men. Preliminary evidence suggests that the smoking effect on C-2 hydroxylation may be opposed by ethanol. Elevated 2-hydroxylation in smokers, in the setting of modestly increased peripheral estrogens and a net decrease in HDLC, may be explained by the fact that lipoprotein synthesis and estrogen 2-hydroxylation both occur predominantly in the liver. Thus, greater metabolic inactivation of hepatic estrogens in male smokers could reduce HDLC, despite a modest rise in circulating hormone levels.

Effects of exogenous thyroxine on C-2 and C-16 alpha hydroxylations of estradiol in humans.

Thyroid dysfunction in humans is known to alter the excretory pattern of estrogen metabolites, suggesting that thyroid hormone directly influences the oxidative metabolism of estradiol. We examined the extent to which a brief period of hyperthyroidism specifically affected estradiol hydroxylation at C-2 and C-16 alpha, the two primary and competing sites of estrogen oxidation, using an in vivo radiometric assay in healthy male volunteers. Hydroxylation at C-2 was increased by a 2-week course of thyroxine (4.3 micrograms/kg/d) from 29.9% +/- 2.6% to 35.9% +/- 3.1% (P = 0.04), while 16 alpha-hydroxylation was unchanged (10.3% +/- 0.8% versus 9.3% +/- 0.5%). The greater extent of oxidation at C-2 was evidenced by a twofold increase in the urinary excretion of 2-hydroxyestrone (2.88 +/- 0.32 versus 5.30 +/- 0.85 micrograms/g creatinine), while the excreted products of 16 alpha-hydroxylation were unchanged. At the same time, significant reductions in total cholesterol (173.8 +/- 7.9 versus 139.4 +/- 8.9 mg/dl), low-density lipoprotein cholesterol (110.0 +/- 5.3 versus 83.8 +/- 7.7 mg/dl), and apolipoprotein B (68.2 +/- 3.3 versus 53.1 +/- 3.6 mg/dl) were observed. Serum levels of estrone, estradiol, sex hormone-binding globulin, high-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, triglycerides, and apolipoprotein A-I were not significantly affected. This study adds to the growing evidence that catechol estrogen production in humans is more readily regulated than 16 alpha-hydroxylation, which is relatively refractory to treatment.

Influence of indole-3-carbinol on the hepatic microsomal formation of catechol estrogens.

The oral administration of indole-3-carbinol (IC), present in cabbage and other members of the Cruciferae family, to female rats almost doubled their ability to convert estradiol to catechol estrogens in the liver. This was determined by the release of 3H from C-2 of the estrogen and also by isolation of the 14C-labeled catechol derivative after incubation with hepatic microsomal fractions. The yield of 4-hydroxyestradiol was also elevated and these effects were similar to those produced by 3-methylcholanthrene (MC), a well-characterized cytochrome P450 inducer. Further evidence for the involvement of a mixed-function oxidase was provided by a 70% to 80% decrease in the yield of 3H2O and water-soluble radioactivity by SKF-525A (0.1 mM) when added to the microsomal fractions isolated from the livers of control or IC-treated rats. In addition, NADPH could not be replaced by NADH in these experiments. Pretreatment with ethionine prevented the increase in estradiol metabolism brought about by oral administration of IC. Both IC and MC inhibited catechol estrogen formation when added directly to the liver microsomal system, confirming earlier findings that in vivo inducers can act as in vitro inhibitors. However, IC was less inhibitory than MC, supporting the theory that IC is converted to a more active product in the stomach. Thus, IC may be conferring protection against estrogen-dependent neoplasia by increasing the hepatic oxidation of estradiol, thereby lowering the amount of available active estrogen.

Increased urinary catechol estrogen excretion in female smokers.

Premenopausal female smokers show significantly increased estrogen 2-hydroxylation, which may account in part for the anti-estrogenic effects of cigarette smoking. We have measured five major urinary estrogens, including estradiol (E2), estrone (E1), 16 alpha-hydroxyestrone (16 alpha OHE1), estriol (E3), and 2-hydroxyestrone (2OHE1), in premenopausal female smokers and non-smokers, to determine whether increased C-2 hydroxylation affected the urinary excretory patterns in these subjects. While total measured estrogen excretion in the follicular phase did not differ significantly between the two groups, urinary 2OHE1 among the smokers constituted a significantly greater proportion of the total (31.1 vs 18.2%, P less than 0.02). This difference was largely caused by significantly increased urinary 2OHE1 and decreased E3 observed in smokers. A urinary catechol estrogen index, defined by [2OHE1]/[E3], was significantly elevated in smokers compared with non-smokers (1.67 +/- 0.21 vs 0.56 +/- 0.08, P less than 0.001), and this urinary index correlated strongly with radiometrically determined estrogen 2-hydroxylation (r = 0.84, P less than 0.01). Ratios of the various estrogen metabolites did not vary substantially throughout the menstrual cycle. Urinary estrogen indices as described here may therefore be useful in demonstrating differences in estrogen metabolism, specifically 2-hydroxylation vs 16 alpha-hydroxylation, in selected populations.

Radioimmunoassay of 16 alpha-hydroxyestrone in human urine.

A radioimmunoassay for the quantitation of the sum of free, glucuronidated and urine is described. The method is reliable and accurate. Using this method, urinary excretion of 16 alpha-hydroxyestrone was determined in normal men, premenopausal women, and postmenopausal women. The values were compared to the urinary excretion of estrone and estradiol. In two women, the urinary excretion of the three estrogens was measured in daily samples throughout a normal menstrual cycle. We conclude that 16 alpha-hydroxyestrone is a quantitatively important urinary estrogen. Inclusion of the measurement of 16 alpha-hydroxyestrone should yield a more accurate assessment of estrogen metabolism.

Increased estrogen 2-hydroxylation in obese women using oral indole-3-carbinol.

OBJECTIVE: To investigate whether the dietary phytochemical, indole-3-carbinol (13C), influences the level of estradiol 2-hydroxylation in obese women. DESIGN: A clinical intervention study involving the ingestion of purified 13C, 400 mg, for two months. SUBJECTS: Five healthy, overweight, premenopausal women (age: 35-47 y, body mass index (BMI): 27-53 kg/m2). MEASUREMENTS: Two estrogen metabolites, 2-hydroxyestrone (2OHE1) and estriol (E3), were measured by radioimmunoassay in untimed overnight urine samples, before and after ingestion of 13C. RESULTS: The ratio of urinary estrogens, 2OHE1/E3, was significantly increased in obese women following 13C, reflecting induction of 2-hydroxylation in these women. CONCLUSIONS: Obese premenopausal women experience increased estrogen 2-hydroxylation in response to the dietary agent, 13C, similar to non-obese women. This response to 13C may result in a hormonal milieu that helps reduce estrogen-dependent cancer risk.

Effects of rapid cerebellectomy on adaptive gain control of the vestibulo-ocular reflex in alert goldfish.

In goldfish, adaptive gain control of the vestibuloocular reflex (VOR) is blocked by cerebellectomy. The operation was rapidly performed on alert goldfish before and after extended periods of adaptive gain training of the VOR produced by sinusoidal oscillation in the horizontal plane. The VOR in these conditions was abolished by sectioning the horizontal semicircular canals. Removal of the cerebellum from naive goldfish resulted in VOR gains significantly greater than 1 at all frequencies tested, with an average value near 1.4 at 1/8 Hz. This value represents an increase of about 65% over the initial VOR gain of 0.85. Changes in phase of the reflex were negligible. Cerebellectomy in animals previously trained to higher or lower gains immediately produced the same mean gain as in cerebellectomized naive animals; gains were increased in animals trained to lower gains and decreased in animals trained to higher gains. As little as 1 min separated aspiration and subsequent gain measurements. These results suggest that the cerebellum not only acts on extra cerebellar circuitry during the training, but that it is also involved in retaining the altered VOR gain. Adaptive gain control could not be achieved with prolonged training after cerebellectomy; in addition, cerebellectomy did not affect the response to visual stimulation at the onset of training to decrease or increase gain.

Altered estrogen metabolism and excretion in humans following consumption of indole-3-carbinol.

Research studies have demonstrated a strong association between estrogen metabolism and the incidence of breast cancer, and we have therefore sought pharmacological means of favorably altering both metabolism and subsequent risk. Indole-3-carbinol (I3C), obtained from cruciferous vegetables (e.g., cabbage, broccoli, etc.), is a known inducer of oxidative P-450 metabolism in animals. We investigated the effects in humans of short-term oral exposure to this compound (6-7 mg/kg/day over 7 days). We used an in vivo radiometric test, which provided a highly specific and reproducible measure of estradiol 2-hydroxylation before and after exposure to I3C. In a group of 12 healthy volunteers, the average extent of reaction increased by approximately 50% during this short exposure (p less than 0.01), affecting men and women equally. We also measured the urinary excretion of two key estrogen metabolites, 2-hydroxyestrone (2OHE1) and estriol (E3). We found that the excretion of 2OHE1 relative to that of E3 was significantly increased by I3C, further confirming the ongoing induction of 2-hydroxylation. These results indicate that I3C predictably alters endogenous estrogen metabolism toward increased catechol estrogen production and may thereby provide a novel "dietary" means for reducing cancer risk.

The effects of cimetidine on the oxidative metabolism of estradiol.

Cimetidine, a histamine H2-receptor antagonist widely used to treat peptic ulceration, is known to cause gynecomastia and sexual dysfunction in some men. Since cimetidine inhibits the cytochrome P-450-dependent biotransformation of numerous drugs, we investigated the possibility that it might also inhibit the cytochrome P-450--dependent metabolism of estradiol. Radiometric analysis of urine and serum samples from nine normal male volunteers showed that the extent of 2-hydroxylation of estradiol was significantly reduced from a mean (+/- SEM) of 31.7 +/- 2.3 percent to 19.7 +/- 2.3 percent (P less than 0.0001) after two weeks of oral treatment with cimetidine (800 mg twice a day); the 16 alpha-hydroxylation of estradiol was unaffected. At the same time, the urinary excretion of 2-hydroxyestrone decreased by approximately 25 percent (P less than 0.0002), and the serum concentration of estradiol increased by approximately 20 percent (P less than 0.04). The mean percentage of estradiol 2-hydroxylation was also rapidly reduced, from 36.8 +/- 4.4 percent to 24.5 +/- 3.4 percent in six men after one week of oral cimetidine at a lower dosage (400 mg twice a day; P less than 0.0006). In a separate study of seven men, ranitidine, a second-generation H2-receptor antagonist, was found to have no effect on the 2-hydroxylation of estradiol. This study demonstrates that the administration of cimetidine to men decreases the 2-hydroxylation of estradiol and results in an increase in the serum estradiol concentration. This mechanism may help to account for the signs and symptoms of estrogen excess reported with the long-term use of cimetidine.

The oxidative metabolism of estradiol: inhibition by cimetidine.

Cimetidine, a histamine H2 antagonist, is known to interfere with the metabolism of exogenous drugs by binding to cytochrome P450. We examined the possibility that cimetidine might also inhibit the cytochrome P450-dependent biotransformation of endogenous compounds such as steroid hormones. Utilizing a radiometric assay and normal male volunteers, the acute effect of intravenous cimetidine (300 mg loading dose followed by 50 mg/hr) was determined. The extent of 2-hydroxylation of estradiol was reduced by 25% from 29.6 +/- 4.4% (mean +/- SEM) before, to 22.9 +/- 4.0% during cimetidine infusion (n = 8; p less than 0.0005). Following oral cimetidine (800 mg b.i.d.) for 2 wk, estradiol 2-hydroxylation was decreased by 40% from 31.7 +/- 2.3% to 19.7 +/- 2.4% (n = 9; p less than 0.0001) but 16 alpha-hydroxylation of estradiol was unaffected. Concomitantly, the urinary excretion of 2-hydroxyestrone was decreased by 25% (p less than 0.002) and the serum estradiol concentration was increased by 20% (p less than 0.04). In contrast, ranitidine, a second generation H2 receptor antagonist, had no effect on estradiol hydroxylation following 150 mg b.i.d. for 2 wk. The inhibition of estradiol 2-hydroxylation and the increase in serum estradiol concentrations caused by cimetidine administration may help to account for the symptoms of hyperestrogenization reported in long-term cimetidine therapy.

Omeprazole fails to alter the cytochrome P450-dependent 2-hydroxylation of estradiol in male volunteers.

Omeprazole, a proton pump inhibitor, is used in the treatment of gastrointestinal diseases associated with hyperacidity. It binds to, and inhibits, some of the activities of hepatic cytochrome P450 resulting in increased half-lives of certain pharmacologic and endogenous compounds. It may also increase the activity of cytochrome P450 under certain conditions. Oxidative metabolism of endogenous estrogens, particularly the 2-hydroxylation pathway, is P450-dependent, and is highly sensitive to a variety of dietary and pharmacologic agents. We therefore studied the extent of estradiol 2-hydroxylation in 7 normal male volunteers before and during oral treatment with omeprazole 20 mg twice daily. Using a specific in vivo radiometric assay, the mean extent (+/- SEM) of estradiol 2-hydroxylation was found to be unchanged before and after omeprazole treatment (27.3 +/- 3.0 vs. 27.5 +/- 3.4%, respectively). The excretion of the endogenous urinary estrogen metabolites, 2-hydroxyestrone, estriol, and estrone was also unaltered by omeprazole. These results show that omeprazole, in contradistinction to other medications used in the treatment of peptic ulcer disease, is without effect on estradiol metabolism in men.

Increased 2-hydroxylation of estradiol as a possible mechanism for the anti-estrogenic effect of cigarette smoking.

Epidemiologic data indicate that cigarette smoking is associated with an important anti-estrogenic effect, and increased hepatic metabolism has been suggested as a possible mechanism. We examined the hypothesis that cigarette smoking in women induces an increase in estradiol 2-hydroxylation. This irreversible metabolic pathway yields 2-hydroxyestrogens, which possess minimal peripheral estrogenic activity and are cleared rapidly from the circulation. We found a significant increase in estradiol 2-hydroxylation in premenopausal women who smoked at least 15 cigarettes per day. The extent of the reaction (mean +/- SEM) was 53.6 +/- 2.2 percent among 14 smokers and 35.1 +/- 1.8 percent among 13 nonsmoking controls--an increase of approximately 50 percent (P less than 0.001). The extent of 2-hydroxylation among five smokers did not vary during the follicular and luteal phases of their menstrual cycles. In addition, urinary excretion of estriol relative to estrone was significantly decreased among smokers (P less than 0.01), providing evidence that the smoking-induced increase in 2-hydroxylation diminishes the competing metabolic pathway involving 16 alpha-hydroxylation. This study demonstrates that smoking exerts a powerful inducing effect on the 2-hydroxylation pathway of estradiol metabolism, which is likely to lead to decreased bioavailability at estrogen target tissues. Elucidation of the mechanism responsible for smoking-induced changes in 2-hydroxylation may be useful in the development of strategies to reduce the risk of hormone-dependent tumors.