Jon J. Michnovicz

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

Abstract 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<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<0.01), provid...

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.

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.

Catechol estrogen production in rat microsomes after treatment with indole-3-carbinol, ascorbigen, or β-naphthaflavone : a comparison of stable isotope dilution gas chromatography-mass spectrometry and radiometric methods

Compounds like indole-3-carbinol (I3C) have been shown to increase catechol estrogen formation and reduce mammary tumor incidence in mice. These compounds may exert a protective effect for breast cancer development by decreasing the overall estrogen pool available for the formation of 16 alpha-hydroxyestrone (16 alpha-OHE1), a metabolite that retains significant estrogenic activity, may be mutagenic and could represent a potential carcinogenic intermediate of estradiol degradation. I3C and ascorbigen originate from the breakdown of glucobrassicin. We have compared the inductive effects of I3C with ascorbigen and beta-naphthaflavone (Bnf) in microsomes from rats pretreated with these compounds using isotope dilution GC-MS and a radiometric method. Incubated microsomes from rats pretreated with I3C and ascorbigen yielded high levels of 2-hydroxyestradiol (2-OHE2) that were comparable to levels induced by Bnf and were significantly above control group levels (p < 0.005). Absolute values determined by the radiometric method were approximately 40% lower than 2-OHE2 concentrations determined by GC-MS, although the relative changes in each group were the same. These differences may be attributed to the radiolabel becoming trapped in microsomal intermediates in the sequence leading to tritium entering the aqueous compartment. Both ascorbigen- and Bnf-treated animals exhibited significant increases in 2-hydroxyestrone (2-OHE1) (p < 0.05). The ability of ascorbigen to induce estradiol C-2 hydroxylation has not been previously reported. Based on these data, we speculate that ascorbigen will act as an anticarcinogenic agent and will inhibit or reduce the incidence of mammary tumor formation.

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).

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.

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.

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)

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.

Radioimmunoassay of 16α-hydroxyestrone in human urine

Abstract 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 16α-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 16α-hydroxyestrone is a quantitatively important urinary estrogen. Inclusion of the measurement of 16α-hydroxyestrone should yield a more accurate assessment of estrogen metabolism.