REPORTS

Urinary 2-Hydroxyestrone/16{alpha}-Hydroxyestrone Ratio and Risk of Breast Cancer in Postmenopausal Women

Giske Ursin, Stephanie London, Frank Z. Stanczyk, Elisabet Gentzschein, Annlia Paganini-Hill, Ronald K. Ross, Malcolm C. Pike

Affiliations of authors: G. Ursin, A. Paganini-Hill, R. K Ross, M. C. Pike, Department of Preventive Medicine, University of Southern California/Norris Comprehensive Cancer Center, Los Angeles; S. London, National Institute of Environmental Health Sciences, Research Triangle Park, NC; F. Z. Stanczyk, E. Gentzschein, Department of Obstetrics and Gynecology, University of Southern California/Los Angeles County Women's Hospital. .

Correspondence to: Giske Ursin, M.D., Ph.D., Department of Preventive Medicine, University of Southern California/Norris Comprehensive Cancer Center, 1441 Eastlake Ave., MS #44, Suite 4407, Los Angeles, CA 90033.


    ABSTRACT
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
BACKGROUND: It has been suggested that women who metabolize a larger proportion of their endogenous estrogen via the 16{alpha}-hydroxylation pathway may be at elevated risk of breast cancer compared with women who metabolize proportionally more estrogen via the 2-hydroxylation pathway. However, the supporting epidemiologic data are scant. Consequently, we compared the ratio of urinary 2-hydroxyestrone (2-OHE1) to 16{alpha}-hydroxyestrone (16{alpha}-OHE1) in postmenopausal women with breast cancer and in healthy control subjects. METHODS: Estrogen metabolites were measured in urine samples obtained from white women who had participated in a previous population-based, breast cancer case-control study at our institution. All P values are from two-sided tests. RESULTS: All of the urinary estrogens measured, with the exception of estriol, were higher in the 66 case patients than in the 76 control subjects. The mean value of urinary 2-OHE1 in case patients was 13.8% (P = .20) higher than that in control subjects, 16{alpha}-OHE1 was 12.1% (P = .23) higher, estrone was 20.9% higher (P = .14), and 17ß-estradiol was 12.0% higher (P = .36). The ratio of 2-OHE1 to 16{alpha}-OHE1 was 1.1% higher in the patients (P = .84), contrary to the hypothesis. Compared with women in the lowest third of the values for the ratio of urinary 2-OHE1 to 16{alpha}-OHE1, women in the highest third were at a nonstatistically significantly increased risk of breast cancer (odds ratio = 1.13; 95% confidence interval = 0.46-2.78), again contrary to the hypothesis. CONCLUSION: This study does not support the hypothesis that the ratio of the two hydroxylated metabolites (2-OHE1/16{alpha}-OHE1) is an important risk factor for breast cancer.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Endogenous ovarian sex steroids play an important role in the etiology of breast cancer (1). The number of premenopausal years (when circulating levels of these hormones are high) is consistently associated with the risk of breast cancer (2). In addition, elevated levels of estrone (E1), 17ß-estradiol (E2), and estriol (E3) have been found in patients with breast cancer compared with control subjects (3).

The main circulating unconjugated estrogen in postmenopausal women is E1, which is readily converted to E2. Two main pathways for metabolizing E2 are via 16{alpha}-hydroxylation and 2-hydroxylation. The major estrogen metabolites are excreted in urine in a conjugated form and include glucuronides and sulfates of the following compounds: 2-hydroxylation products (2-hydroxyestrone [2-OHE1], 2-hydroxyestradiol, and 2-methoxyestrone), 16{alpha}-hydroxylation products (E3 and 16{alpha}-hydroxyestrone [16{alpha}-OHE1]), E1, and E2 (4). The roles of these estrogen metabolites have been the subject of much discussion. The 16{alpha}-hydroxylated metabolites are biologically active (5,6). The extent to which the 2-hydroxylated metabolites are active or can form active or genotoxic metabolites is controversial (7-10). However, some investigators characterize 2-OHE1 as the "good estrogen" (11) and have suggested that the amount of estrogen that is metabolized via the 16{alpha}-hydroxylation pathway may be associated with the risk of developing breast cancer (12-14).

Epidemiologic data for this hypothesis are sparse. Two small studies found that the extent of 16{alpha}-hydroxylation was higher in women with breast cancer (15) and in women with a high familial risk of breast cancer (16) than in control women. However, a third study (4) found no elevation of 16{alpha}-hydroxylation products in patients with breast cancer compared with control subjects. A fourth study (17) found no important association between the ratio of 2-OHE1 to 16{alpha}-OHE1 and breast cancer when premenopausal and postmenopausal women were considered together, but it found a strong inverse association when data from the 23 postmenopausal patients and 28 postmenopausal control subjects were compared. The control subjects in all of these studies were simply convenience samples. A recent nested case-control study (18) reported an inverse association, but the findings were not statistically significant and the confidence intervals (CIs) were wide.

We collected urine samples from participants in a previously conducted, population-based, case-control study to determine whether postmenopausal women with breast cancer have a lower ratio of urinary 2-OHE1/16{alpha}-OHE1 than control subjects, as proposed by the "good estrogen" theory. Previously, we (19) have reported unadjusted pilot results from the first 25 patients and 23 control subjects that used an earlier version of the 2-OHE1 and 16{alpha}-OHE1 assay.

In this report, we present results from 66 patients and 76 control subjects. All samples were analyzed with an updated version of the 2-OHE1 and 16{alpha}-OHE1 assay.


    SUBJECTS AND METHODS
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Subjects

We included patients with breast cancer and control subjects who had previously participated in a population-based, case-control study at the University of Southern California. Patients were women with a diagnosis of breast cancer identified through the Los Angeles County Cancer Surveillance Program (the Los Angeles National Cancer Institute Surveillance, Epidemiology, and End Results [SEER]1 registry) who were English-speaking residents of Los Angeles County. Patients were diagnosed with breast cancer from March 1, 1987, through December 31, 1989 (20), or from January 1, 1992, through December 31, 1992, were white (including Latina), and were born in the United States, Canada, or Western Europe from 1923 through 1937. Control women were individually matched to patients by age (±3 years), ethnicity (Latina/non-Latina), and neighborhood of residence. The response rates in the first part of the study were 64% for patients and 80% for control subjects (20). Written informed consent was obtained from all participants. In this estrogen metabolism study, we targeted a subset of the patients and control subjects from the parent study. We included only patients diagnosed with incident cancer localized to the breast as defined by the SEER summary staging (21) (tumor of any size, confined to breast tissue and fat, and no distant metastasis). Additional eligibility criteria were obtained from published data and discussions with experts in the field of 2-hydroxy and 16{alpha}-hydroxy estrogen metabolism and are as follows: 1) no medications that might interfere with estrogen metabolism within the previous 6 months (specifically, cimetidine, thyroid supplements, estrogen or progesterone compounds, tamoxifen, or {omega}-3 fatty acid supplements); 2) never used cancer chemotherapeutic agents (22-25); 3) no general anesthesia in the previous 3 months; 4) weight between 80 and 200 pounds (36 and 90 kg); and 5) nonsmoker for the past 3 years.

On the basis of data from the parent study, we identified 458 nonsmoking patients who weighed less than 200 pounds and 732 corresponding control subjects. We attempted to contact all of these women by telephone and/or letter to determine eligibility for this study. We were unable to contact 51 patients and 93 control subjects because they had moved and no forwarding address was available. An additional 19 patients and one control subject had died. Of the remaining 388 patients and 638 control subjects, we successfully contacted 312 (312/458 = 68.1%) patients and 329 (329/732 = 44.9%) control subjects. A large number of responders (211 patients and 220 control subjects) were found to be ineligible because of recent medication use (tamoxifen [144 patients], thyroid supplements [22 patients and 17 control subjects], chemotherapy or megestrol acetate [17 patients], estrogen [17 patients and 168 control subjects], or other medication [14 patients and 31 control subjects]), current weight of more than 200 pounds (nine patients and nine control subjects), recent general anesthesia (one patient and one control subject), or currently smoking (one patient). Fourteen patients and six control subjects met two exclusion criteria. Twenty-five eligible patients and 24 eligible control subjects refused to participate. In addition, we were unable to schedule urine collection for two patients before the study ended. We enrolled 74 patients and 85 control subjects. We subsequently excluded two additional patients and four additional control subjects because of incomplete risk factor information, one patient with in situ breast cancer, two patients with unknown disease stage, and three patients and five control subjects whose samples had very low urinary creatinine levels (<0.20 mg/mL). We present the results on the remaining 66 patients and 76 control subjects.

To each participant, we shipped a box containing a 100-mL urine vial with a 100-mg ascorbate tablet, a small cooler with an ice pack, an informed consent form, and a questionnaire on recent intake of medications, alcohol, and specific foods. We used a semiquantitative food-frequency questionnaire on their usual diet over the past 12 months (26,27) and asked the women to report their intake of alcohol and cruciferous vegetables during the 24- and 48-hour periods, respectively, before urine collection. The first morning urine samples were returned to us. All samples were frozen at -70 °C within 30 hours after collection. Samples, blinded for the case or control status, were shipped to two laboratories for analyses (see below). The urine collection was conducted from 1993 through 1996. Levels of estrogen metabolites did not differ by time since diagnosis in patients (data not shown).

Enzyme Immunoassay of 16{alpha}-OHE1 and 2-OHE1

Measurements of urinary 16{alpha}-OHE1 and 2-OHE1 were carried out in the laboratories of H. L. Bradlow and D. Sepkovicz (Strang Cornell Cancer Research Laboratory, New York, NY). Commercially available competitive enzyme immunoassay kits (Estramet; Immuna Care Corporation, Bethlehem, PA) were used to measure 2-OHE1 and 16{alpha}-OHE1 directly in urine. High-affinity murine monoclonal antibodies specific for the estrogen metabolites were immobilized directly on microtiter plates, and the enzyme alkaline phosphatase was linked to each estrogen metabolite, which was used to compete with a standard or analyte in the assay. Each sample of urine was acidified and subjected to ß-glucuronidase/aryl sulfatase hydrolysis before it was assayed (28). These kits have been validated previously (28,29). Samples that had been analyzed with a previous version of the assay (19) were assayed again with the adjusted version that allowed for the lower estrogen values among postmenopausal women. The intra-assay coefficients of variation were 15.7% for 2-OHE1 and 16.2% for 16{alpha}-OHE1.

Radioimmunoassay of Urinary E1, E2, and E3

Measurements of urinary E1, E2, and E3 were carried out in the laboratory of F. Z. Stanczyk (Los Angeles County/University of Southern California Medical Center) with high-performance liquid chromatography and radioimmunoassays (19). Each sample of urine was acidified and subjected to ß-glucuronidase/aryl sulfatase hydrolysis before it was assayed. The E1, E2, and E3 fractions were quantified by radioimmunoassay as described previously (30-32). Quality-control samples were included with each set of samples assayed to monitor assay reliability. The intra-assay coefficients of variation were 9.3% for E1, 16.8% for E2, and 13.6% for E3.

Statistical Analysis

All of the directly measured hormone variables were logarithmically transformed. The statistical significance of the difference in these variables between patients and control subjects was thus evaluated by use of t tests on the natural logarithms. We used logistic regression to determine the odds ratio (OR) of breast cancer associated with various ratios of urinary 2-OHE1/16{alpha}-OHE1 and computed P values for Mantel's test for trend (33). All P values are from two-sided tests. Because this study did not maintain the matching of the original enrollment, we used unconditional logistic regression but adjusted for the original matching variables [age, ethnicity, and socioeconomic status based on area of residence (34)] and other potential confounders (33).


    RESULTS
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Data on 66 patients and 76 control subjects were available for this study. Table 1Go shows the ORs associated with selected risk factors in this population. As expected, the highest risk of breast cancer was observed for heavier women, women of high socioeconomic status, women with no children, and women with early menarche. The parity finding was the only statistically significant observation.


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Table 1. Odds ratios (ORs) and 95% confidence intervals (Cis) for postmenopausal breast cancer associated with selected factors

 
The geometric mean levels of urinary estrogen metabolites are shown in Table 2.Go All estrogens except E3 were higher in patients than in control subjects. Although none of these differences was statistically significant, the mean value for 2-OHE1 was 13.8% higher and the mean value for 16{alpha}-OHE1 was 12.1% higher in patients than in control subjects. The ratios of 2-OHE1/16{alpha}-OHE1 were essentially identical.


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Table 2. Geometric mean levels of urinary estrogen metabolites in postmenopausal patients with breast cancer and in control subjects

 
Table 3Go shows the ORs of breast cancer associated with various categories of the ratio 2-OHE1/16{alpha}-OHE1. There was no suggestion that an increased ratio of 2-OHE1/16{alpha}-OHE1 was associated with a decreased risk of breast cancer.


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Table 3. Odds ratios (ORs) and 95% confidence intervals (CIs) for postmenopausal patients with breast cancer according to tertile categories of urinary estrogen metabolites

 
These results were unchanged when they were adjusted for usual intake of fat, animal fat, vegetable fat, protein, total calories, or calories from fat. When adjusted for intake of alcohol and cruciferous vegetables during the 24 hours preceding urinary collection, the OR estimates for all estrogens increased between 5% and 20%. However, the adjusted ORs for the ratio 2-OHE1/16{alpha}-OHE1 (0.32 for the second tertile and 1.14 for the third tertile) were almost identical to those presented in Table 3.

Table 4Go shows the geometric mean levels for 2-OHE1, 16{alpha}-OHE1, and the ratio 2-OHE1/16{alpha}-OHE1 by selected risk factors. There were no remarkable differences in the estrogen metabolite ratios across the levels of these risk factors; all of the CIs overlapped.


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Table 4. Geometric mean levels (95% confidence intervals [CIs]) of urinary estrogen metabolites (µg/g of creatinine) in postmenopausal control subjects by selected risk factors*

 

    DISCUSSION
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The data from this study do not support the hypothesis that the ratio of the two hydroxylated metabolites (2-OHE1/16{alpha}-OHE1) is an important risk factor for breast cancer (12-14). In our study, these ratios were essentially identical in patients and control subjects. Apart from E3, all of the urinary estrogens measured in this study were higher in patients with breast cancer than in control subjects. These results confirm previous studies (35-42).

Epidemiologic data addressing the 2-OHE1/16{alpha}-OHE1 hypothesis are sparse. Schneider et al. (15) injected 33 perimenopausal and postmenopausal breast cancer patients and 10 postmenopausal control subjects with E2 labeled with tritium in the 17{alpha}, C2, and 16{alpha} positions. They determined the extent of oxidative metabolism at the different sites by drawing serial blood samples after the isotope administration. Patients had 60% greater 16{alpha}-hydroxylation than did control subjects; this difference was statistically significant. However, the two groups did not differ in a statistically significantly fashion in 2-hydroxylation, which was 5% higher in patients. The ratio of the average level of 2-hydroxylation to the average level of 16{alpha}-hydroxylation was 52% greater in the control subjects than in the patients with breast cancer. Adlercreutz et al. (4) examined estrogen metabolites in 10 young Finnish premenopausal patients with breast cancer, 12 control women who were omnivorous, and 11 control women who were lacto-vegetarians. There was no statistically significant difference in 2-OHE1 or 16{alpha}-OHE1 for these groups. In another study, Adlercreutz et al. (43) reported that the ratio of 2-hydroxylation products to 16{alpha}-hydroxylation products was fourfold to fivefold higher among premenopausal Finnish women at high risk for breast cancer than among 13 premenopausal Asian women, a low-risk population, contrary to the hypothesized result (12-14).

Kabat et al. (17) obtained spot urine samples from 42 patients with breast cancer and 64 women attending a free breast cancer screening clinic. They found no differences in the ratio 2-OHE1/16{alpha}OHE1 between these patients and control subjects. However, they reported a statistically significantly lower ratio in the 23 patients than in the 28 control subjects who were postmenopausal. There was no weight restriction in that study; thus, it is conceivable that the impressive results in postmenopausal women might have been due to inclusion of obese postmenopausal patients with breast cancer, who, because of their obesity, have abnormally low levels of 2-hydroxylation products (44). Furthermore, the majority of the patients with breast cancer in this study had recently undergone surgery. Compounds with a substantial hepatic metabolism may reduce 2-hydroxylation (23,45); the effect that barbiturates administered as part of general anesthesia may have on 2-hydroxylation is unknown.

Recently, Meilahn et al. (18) conducted a nested case-control study within the Guernsey III cohort follow-up. They reported a median for the ratio 2-OHE1/16{alpha}-OHE1 of 1.6 for the 42 postmenopausal patients and of 1.7 for the 139 matched control subjects. Compared with the postmenopausal women who had values for the ratio 2-OHE1/16{alpha}-OHE1 in the lowest third, women who had values in the highest third had an OR for breast cancer of 0.71, but the CI was wide and not statistically significant (95% CI = 0.29-1.75).

Experimental studies lend some support to the hypothesis of a protective effect for a high 2-OHE1/16{alpha}-OHE1 ratio. In vitro data suggest that 16{alpha}-OHE1 induces a higher rate of cell proliferation than 2-OHE1 (46,47) and that 16{alpha}-OHE1 induces tumors in hamsters (48). Furthermore, the extent of [3H]E2 metabolism via the 16{alpha}-hydroxylation pathway was reported to be 4.6-fold higher in terminal duct lobular units in breast tissue from patients with breast cancer than in breast tissue from control subjects who underwent reduction mammoplasty (49).

However, not all data are supportive. In fact, some investigators (50) found that 2-hydroxylated metabolites stimulate proliferation in MCF-7 cells. Furthermore, there is evidence that catechol estrogens can cause DNA damage directly by forming quinones and semiquinones and DNA damage indirectly through the production of radical intermediates during metabolic redox cycling between catechol and quinone forms (10).

Our results, although clearly not supportive of this hypothesis, may have been affected by several factors. We purposely studied a select group of women with few extraneous factors that might influence estrogen metabolism but excluded a large number of women. However, the exclusions were applied equally to patients and control subjects, and none of these exclusions appears likely to introduce any biases in one direction or the other.

Despite our many attempts to contact patients and control subjects, we cannot exclude the possibility that women with a healthy lifestyle (and a high 2-OHE1/16{alpha}-OHE1 ratio) may have been more likely to participate in this study than other women. However, we would expect this to be more frequent among control subjects. If so, one would expect any bias to favor the hypothesis of Bradlow et al. (12-14).

We studied patients with breast cancer who were diagnosed and had been treated for early stage breast cancer 3-7 years earlier. It is not known whether the onset of cancer and its sequelae may affect 2-hydroxylation and 16{alpha}-hydroxylation. If so, then this would be a limitation in any case-control test of the 2-OHE1/16{alpha}-OHE1 hypothesis that collected urine samples after the disease process had started.

Certain medications, including tamoxifen, influence the hydroxylation pattern (22-25); therefore, we excluded all women who had ever been treated with chemotherapy or who had recently used tamoxifen. Because our study took place several years after breast cancer surgery, we think it is unlikely that the primary treatment for breast cancer affected the hydroxylation pattern among the patients.

Whether survival may be poorer in patients with breast cancer who have a lower 2-OHE1/16{alpha}-OHE1 ratio is unknown. However, we only studied patients with early stage disease for whom survival is high; only 4.1% had died.

The evidence is rather clear that certain diets influence the extent of 16{alpha}-hydroxylation and 2-hydroxylation (45,51-53). However, adjustments for usual intake of macronutrients and recent intake of cruciferous vegetables or alcohol did not change the results. Rather than using a 24-hour urine collection, we used a first morning void urine sample and adjusted for creatinine. Other investigators (17) have used similar approaches and have obtained positive results.

In contrast to most previous studies, our study was based on a population-based, case-control study, not a convenience sample of patients and control subjects. It was able to demonstrate effects as large as those observed by Kabat et al. (17) with very high statistical power. However, our results from this case-control study of breast cancer in postmenopausal women support no association between the ratio of urinary 2-OHE1 to 16{alpha}-OHE1 and the risk of breast cancer.


    NOTES
 
1 Editor's note: SEER is a set of geographically defined, population-based, central cancer registries in the United States, operated by local non profit organizations under contract to the National Cancer Institute (NCI). Registry data are submitted electronically to the NCI on a biannual basis, and the NCI makes the data available for analysis. Back

Supported by grants DAMD17-94-J-4289 and DAMD17-94-J-4049 (G. Ursin from the U.S. Army Medical Research and Materiel Command; by Public Health Service grant P01CA17054 (G. Ursin) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; and by the California Department of Health Services, through the California Public Health Foundation, as part of its statewide cancer reporting program, mandated by Health and Safety Code Sections 210 and 211.3. G. Ursin and S. London were supported in part by Research Career Development Awards from the Stop Cancer Foundation.

The ideas and opinions expressed herein are those of the authors, and no endorsement by the State a California Department of Health Services or the California Public Health Foundation is intended or should be inferred.


    REFERENCES
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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Manuscript received November 19, 1999; revised April 7, 1999; accepted April 15, 1999.


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