Published OnlineFirst July 26, 2018; DOI: 10.1158/1055-9965.EPI-18-0135
Research Article Cancer Epidemiology, Biomarkers Estrogen Metabolism in Postmenopausal Women & Prevention Exposed In Utero to Diethylstilbestrol Rebecca Troisi1, Elizabeth E. Hatch2, Julie R. Palmer3, Linda Titus4, Joshua N. Sampson1, Xia Xu5, and Robert N. Hoover1
Abstract
Background: Prenatal diethylstilbestrol (DES) exposure is Results: Concentrations of total estrogens (15.3%; CI, associated with adverse reproductive outcomes and cancer of 4.1–38.5) and parent estrogens (27.1%; CI, 8.2–76.1) the breast and vagina/cervix in adult women. DES effects on were slightly higher in the DES-exposed than unexposed. estrogen metabolism have been hypothesized, but reproduc- Ratios of path2:parent estrogens ( 36.5%; CI, 53.0 to tive hormone concentrations and metabolic pathways have 14.3) and path2:path16 ( 28.8%; CI, 47.3–3.7) were not been comprehensively described. lower in the DES exposed. These associations persisted Methods: Blood samples were provided by 60 postmeno- with adjustment for total estrogen, years since meno- pausal women (40 exposed and 20 unexposed) who were pause, body mass index, parity, and recent alcohol participants in the NCI Combined DES Cohort Study, had intake. never used hormone supplements or been diagnosed with Conclusions: These preliminary data suggest that postmen- cancer, had responded to the most recent cohort study ques- opausal women who were prenatally DES exposed may have tionnaire, and lived within driving distance of Boston Uni- relatively less 2 than 16 pathway estrogen metabolism com- versity Medical School (Boston, MA). Parent estrogens and pared with unexposed women. their metabolites were measured by high-performance liquid Impact: Lower 2 pathway metabolism has been associat- chromatography–tandem mass spectrometry. Age-adjusted ed with increased postmenopausal breast cancer risk and percent changes in geometric means and associated 95% could potentially offer a partial explanation for the modest confidence intervals (CIs) between the exposed and unex- increased risk observed for prenatally DES-exposed women. posed were calculated. Cancer Epidemiol Biomarkers Prev; 27(10); 1208–13. 2018 AACR.
Introduction be responsible for the adverse health outcomes observed in exposed individuals. The synthetic estrogen diethylstilbestrol (DES), to which mil- In humans, the hormones estradiol and estrone when lions were exposed, is the only known human transplacental hydroxylated at the 2-, 4-, or 16-carbon position, result in an carcinogen, as in utero exposure has been linked with cancers of array of metabolites that have varying carcinogenicity. Recent the vagina (1) and breast (2), as well as cervical intraepithelial observations in humans show that breast cancer risk is related neoplasia grade 2 and higher (3). Environmental scientists also to specific metabolic pathways, with greater metabolism in the consider DES the definitive model for the impact of exposure to 2 pathway showing protection (6). This led us to assess whether environmental endocrine disruptors during fetal development hormones and their metabolite concentrations were altered in (4). More than three decades of studies in laboratory animals have postmenopausal women with documented prenatal exposure raised multiple possible biologic mechanisms, including a pos- to DES. sible influence of DES on hormone concentrations (5) that could Materials and Methods 1 Division of Cancer Epidemiology and Genetics, Department of Health and The data for the current analysis were collected as part of a Human Services, NCI, NIH, Bethesda, Maryland. 2Department of Epidemiology, feasibility study at Boston University (Boston, Massachusetts), 1 Boston University School of Public Health, Boston, Massachusetts. 3Slone Epidemiology Center and BU-BMC Cancer Center, Boston University, Boston, of 5 sites of the NCI's Combined DES Cohort Follow-up Study. Massachusetts. 4Departments of Epidemiology and Pediatrics, Geisel School of The study was approved by institutional review boards at the NCI Medicine at Dartmouth, the Norris Cotton Cancer Center, and the Hood Center and Boston University. for Children and Families, Lebanon, New Hampshire. 5Cancer Research Tech- nology Program, Leidos Biomedical Research, Inc., Frederick National Labora- NCI's combined DES cohort follow-up study tory for Cancer Research, Frederick, Maryland. In 1992, the NCI assembled extant U.S. cohorts of individuals Note: Supplementary data for this article are available at Cancer Epidemiology, with medical record documentation of exposure or lack of expo- Biomarkers & Prevention Online (http://cebp.aacrjournals.org/). sure to DES, including daughters identified from the National Corresponding Author: Rebecca Troisi, DCEG/NCI/NIH, MSC 9773, 9609 Cooperative Diethylstilbestrol Adenosis Project (DESAD; ref. 7), Medical Center Drive, Rockville, MD 20850. Phone: 240-276-6385, E-mail: and a large private infertility practice in Massachusetts. A cohort of [email protected] daughters of women who participated in the Women's Health doi: 10.1158/1055-9965.EPI-18-0135 Study [(WHS) Daughters' Cohort; ref. 8] was added to the com- 2018 American Association for Cancer Research. bined cohort in 1994. Questionnaires were mailed in 1994, and
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approximately every 5 years since to ascertain information on 4-methoxyestrone, 4-methoxyestradiol, 16a-hydroxyestrone, health outcomes and major cancer risk factors. A detailed descrip- 16-ketoestradiol, estriol, 17-epiestriol, and 16-epiestriol, in serum tion of the methods and findings from the main cohort study is as described previously (13, 14) with updated instrumentations published (9). and additional stable isotope–labeled estrogen metabolites. Brief- ly, HPLC-MS/MS analysis was performed using a Thermo TSQ Substudy Vantage triple quadrupole mass spectrometer (Thermo Fisher We sought to enroll 40 exposed and 20 unexposed women. Scientific) coupled with a Prominence LC system (Shimadzu We restricted eligibility for this substudy to women who had Scientific Instruments). Both HPLC and MS were controlled by responded to the most recent study questionnaire (2011) and Xcalibur software (Thermo Fisher Scientific). Twelve stable iso- lived within driving distance of the Boston University Medical topically labeled estrogens and estrogen metabolites were used to School General Clinical Research Unit (GCRU). We further account for losses during sample preparation and HPLC-MS/MS restricted to postmenopausal women who had never used analyses, which included deuterated estriol, (C/D/N Isotopes, hormone supplements and had never been diagnosed with Inc.); deuterated 16-epiestriol (Medical Isotopes, Inc.); and any cancer (excluding nonmelanoma skin cancer). History of 13C-labeled estrone, estradiol, 2-hydroxyestrone, 2-methoxyes- hormone therapy use was based on any questionnaire report of trone, 2-hydroxyestradiol, 2-methoxyestradiol, 2-hydroxyes- use of oral medications, vaginal cream/tablet/ring, and topical trone-3-methyl ether, 4-hydroxyestrone, 4-methoxyestrone, and creams. At the time of blood draw, women were also asked 4-methoxyestradiol (Cambridge Isotope Laboratories). Measure- about current and recent use in case they began using hormones ments were made at the Laboratory of Proteomics and Analytical since their last questionnaire report. Of 2,407 Boston Univer- Technologies, Cancer Research Technology Program, Leidos Bio- sity participants, 793 women lived within 30 miles and among medical Research, Inc. The stable isotope dilution HPLC-MS/MS them, 300 met the other inclusion criteria. We also attempted was used to quantify 15 estrogens and estrogen metabolites that to choose women with vaginal epithelial changes (VEC), which circulate primarily as sulfated and/or glucuronidated conjugates are correlated with a higher dose of, and earlier exposure to, (14). Five (estrone, estradiol, estriol, 2-methoxyestrone, and DES in utero (10). We wrote to 180 of the 300 eligible women 2-methoxyestradiol) were measured in unconjugated forms. The inviting them to participate in data collection and then fol- serum sample was split into two aliquots to measure the com- lowed up with a phone call: 16 were found to be ineligible due bined concentration of each of the 15 estrogens (sum of conju- to(i)havingmovedoutofthearea(n ¼ 4), (ii) death (n ¼ 1), gated plus unconjugated forms) and the unconjugated forms. To or (iii) not meeting other eligibility criteria (n ¼ 11). Of 164 measure the combined parent estrogen or estrogen metabolite eligible women, 44 refused (27%), 60 (37%) were either not level, an enzymatic hydrolysis with sulfatase and glucuronidase called or not reached by telephone, and 60 agreed to partici- activity was added to the sample preparation to cleave any sulfate pate. The 60 participants (40 exposed, 20 unexposed) provided and glucuronide groups. To measure the unconjugated forms, the blood between May 2014 and July 2015. enzymatic hydrolysis was not included in sample preparation. For Participants gave written informed consent, completed a metabolites with both combined and unconjugated measure- short questionnaire that confirmed their eligibility and ascer- ments, the concentration of the conjugated form was calculated tained recent alcohol use, and had their height and weight as the difference between the combined and unconjugated estro- measured. Body mass index (BMI) was calculated by dividing gen measurements. The calibration curves were linear over a weight (kg) by height (m; ref. 2). Three 10 mL tubes of whole concentration range of 1 pg/mL to 1,000 pg/mL for all estrogens blood were collected and immediately processed. Serum sam- and estrogen metabolites. The assay limit of detection (LOD) ples were stored in the GCRU and later shipped on dry ice to the providing estrogen signal-to-noise ratio greater than 3-to-1 was NCI's laboratory. 100 fg/mL ( 0.33–0.37 pmol/L). The assay lower limit of quan- The DESAD study incorporated a comprehensive gynecologic titation (LLOQ) was 1 pg/mL for each estrogen and estrogen examination around the time of recruitment that systematically metabolite with intra- and interbatch coefficients of variation identified VEC by means of colposcopy or iodine staining <15% and assay accuracy between 85% and 115% of known (11, 12). Vaginal epithelial changes were defined as vaginal targeted values at LLOQ. Laboratory coefficients of variation from epithelium that was glandular in nature (adenosis) or metaplastic 10 blinded replicates across batches were <3.0% for all hormones squamous epithelium, which develops as adenosis undergoes measured. physiologic healing. Age at menarche was ascertained on the Estrogens and estrogen metabolites were analyzed individual- 1994 questionnaire, parity and smoking status were ascertained ly, in groups representing metabolic pathways, and as ratios of on the 2006 questionnaire, and age at menopause was queried on metabolic pathways. Estrone and estradiol, the parent estrogens, all of the questionnaires. On the basis of knowledge of regional are irreversibly hydroxylated at the C-2, C-4, or C-16 positions of prescribing practices, we characterized Boston's cohorts as "high the steroid ring, leading to a cascade of metabolites (Supplementary dose" (2). Among participants with complete information on Fig. S1). The combined metabolites in each of the C-2, C-4, and C- cumulative dose, the median dose for Boston was 8,675 mg. 16 pathways were used to investigate whether prenatal DES expo- sure was associated with altered patterns of estrogen metabolism. Estrogen assay These included metabolites in the 2-hydroxylation pathway (Path2, A validated and reported stable isotope dilution high- i.e., 2-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestradiol, 2- performance liquid chromatography–tandem mass spectrometry methoxyestradiol, and 2-hydroxyestrone-3-methyl ether); metabo- (HPLC-MS/MS) assay was employed to measure 15 estrogens lites in the 4-hydroxylation pathway (Path4, i.e., 4-hydroxyestrone, and estrogen metabolites, including estrone, estradiol, 2-hydro- 4-methoxyestrone, and 4-methoxyestradiol); and metabolites in xyestrone, 2-methoxyestrone, 2-hydroxyestradiol, 2-methoxyes- the 16-hydroxylation pathway (Path16, i.e., 16a-hydroxyestrone, tradiol, 2-hydroxyestrone-3-methyl ether, 4-hydroxyestrone, estriol, 17-epiestriol, 16-ketoestradiol, and 16-epiestriol). These
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pathways were also examined as ratios—for example, path2: ment) in SAS. In separate models, we further adjusted for years path16—to assess relative metabolism down each of the path- since menopause, BMI, parity, and recent alcohol use. Forest plots ways. Total parent estrogens were the sum of estrone and estra- were created to visually present the percent change in the means diol; total metabolites were the sum of Path2, Path4, and Path16; for the parent estrogens and metabolites between the exposed and and total estrogens were defined as the sum of the parent estrogens unexposed with 95% confidence intervals (CIs). and metabolites. Results Statistical analysis Values for the estrogens were examined for outliers and those Mean age was 60.8 years (range, 51.6–67.7) for exposed wom- with 1.5 interquartile ranges above the 75% percentile or below en and 61.7 years (55.9–67.8) for unexposed women, and all the 25% percentile were excluded for the analysis of that estrogen. participants identified themselves as white. Reproductive, anthro- Values were log transformed to improve normality. Pearson pometric, and lifestyle characteristics are presented in Table 1. The correlations were calculated among the 15 estrogens. Standard exposed and unexposed women were generally similar in these linear models (Proc GLM; SAS) with log estrogen (or log ratio) as characteristics. Thirty-six of the 40 exposed women had a history the dependent variable and DES status as the independent var- of VEC. iable evaluated the effect of DES exposure. Models were adjusted In the exposed and unexposed combined, parent estrogens, for age (continuous variable). The coefficient, b, for DES was Path2, Path4, and Path16 metabolites comprised 38.3% exponentiated and estimates the ratio of the geometric means (estrone 34.6% and estradiol 3.7%), 16.3%, 1.7%, and between the two groups. The percentage change is defined as 43.6% of total estrogens (sum of all 15 concentrations), respec- 100 (exp(b)-1). A positive percent change indicates that the tively (Supplementary Fig. S2). The parent hormones, estrone mean is higher in the DES exposed compared with the unexposed, and estradiol, were highly correlated (r ¼ 0.84), whereas the and a negative percent change indicates that the mean is lower other metabolites showed less correlation (Supplementary in the exposed compared with the unexposed. The reported age- Table S1). adjusted geometric means for each of the DES groups were Age-adjusted geometric mean serum concentrations of the estimated using the least-square means procedure (lsmeans state- parent estrogens and estrogen metabolites by prenatal DES
Table 1. Characteristics of participants by prenatal DES exposure DES exposed DES unexposed n ¼ 40 % n ¼ 20 % Birth year <1950 8 20.0 5 25.0 1950–1954 17 42.5 8 40.0 1955þ 15 37.5 7 35.0 Birth year (continuous) 1953.5 1947–1962 1952.8 1947–1959 Age at menarche 14þ 10 25.0 2 10.0 12–13 24 60.0 13 65.0 11 6 15.0 5 25.0 Age at menarche (continuous) 12.7 9–15 12.5 11–16 Age at menopause <50 7 17.5 4 20.0 50–54 29 72.5 14 70.0 55–59 4 10.0 2 10.0 Age at menopause (continuous) 50.9 40–57 51.5 47–57 Years since menopause 10 21 52.5 9 45.0 10–19 17 42.5 11 55.0 20–29 2 5.0 0 0 Years since menopause (continuous) 9.9 4–27 10.3 3–19 BMI at blood draw 20 0 0 3 15.0 20–24 10 25.0 4 20.0 25–29 12 30.0 5 25.0 30þ 18 45.0 8 40.0 BMI at blood draw (continuous) 30.0 20–55 28.3 18–47 Parous (2006 questionnaire) No 15 37.5 10 50.0 Yes 25 62.5 10 50.0 Ever smoke (2006 questionnaire) Yes 19 47.5 9 45.0 No 21 52.5 11 55.0 Alcohol in last 24 hours No 26 65.0 17 85.0 Yes 14 35.0 3 15.0 NOTE: Categorical variables are number and percent; continuous variables are mean and range.
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Table 2. Age-adjusted geometric means (95% CI)a for parent estrogens and their metabolites by prenatal DES exposure DES exposed (n ¼ 40) DES unexposed (n ¼ 20) Estrogen/EM (pmol/L) Mean 95% CI SE Mean 95% CI SE % Change P Total estrogens 930.9 834.3–1039 50.9 807.5 694.1–939.6 61.0 15.3 0.13 Parent estrogens 340.5 282.1–410.9 32.0 267.8 203.7–352.0 36.6 27.1 0.15 Estrone 305.2 251.4–370.6 29.6 236.5 178.4–313.7 33.3 29.0 0.14 Estradiol 33.5 28.6–39.3 2.64 27.04 21.2–34.5 3.29 23.9 0.15 Total metabolites 552.1 496.1–614.3 29.4 530.3 456.6–615.9 39.6 4.11 0.66 Path2 144.4 131.6–158.5 6.7 166.1 145.6–189.5 10.9 13.1 0.09 Path4 15.4 13.3–18.0 1.18 15.2 12.2–18.8 1.64 1.76 0.90 Path16 379.5 327.1–440.2 28.1 325.5 264.3–400.8 33.8 16.6 0.24 Total metabolites: parent estrogens 1.68 1.43–1.97 0.13 1.97 1.56–2.49 0.23 15.1 0.26 Path2: parent estrogens 0.42 0.36–0.50 0.03 0.67 0.51–0.86 0.09 36.5 0.005 Path4: parent estrogens 0.05 0.04–0.06 0.006 0.06 0.04–0.09 0.01 25.6 0.19 Path16: parent estrogens 1.16 0.96–1.41 0.11 1.30 0.99–1.71 0.18 10.8 0.50 Path2: Path16 0.37 0.31–0.44 0.03 0.51 0.40–0.66 0.06 28.8 0.03 Path4: Path16 0.04 0.03–0.05 0.004 0.05 0.04–0.06 0.007 16.7 0.32 Path2: Path4 9.36 7.96–11.0 0.75 11.0 8.71–13.8 1.25 14.6 0.27 aMeans and SE are presented exponentiated from the logarithmic scale.
exposure are presented in Table 2. In general, total estrogens of the epigenetic regulators DNA methyltransferase (DNMT) 1 and parent estrogens were greater in the DES-exposed women. and DNMT3A in the juvenile cortex and hypothalamus, par- The ratios of path2:total estrogens, path2:parent estrogens, alleling changes in estrogen-related receptors. Importantly, and path2:path16 (i.e., less metabolism in the 2 vs. 16 path- changes in ERa and DNMT expression in the female hypothal- way) were all lower in the DES-exposed women, whereas the amus were associated with DNA methylation changes in the ratio of path16:total metabolites was greater in the DES ERa gene. There are no direct data in animals on the effect of exposed. Supplementary Figure S3 shows the same informa- DES on estrogen metabolism. tion presented in Table 2, age-adjusted percent change in Data on the effect of prenatal DES exposure on hormone geometric means between the prenatally DES exposed and the concentrations in humans are scant. Two small studies of DES unexposed in parent estrogens, estrogen metabolites, metab- and hormones (16, 17) were conducted in the 1970s with olite pathways, and ratios. Associations were similar with inconsistent results and usinghormoneassaymethodsfar additional adjustment for years since menopause, BMI, parity, inferior to those now available. More recently, data from a and recent alcohol intake (Fig. 1). small sample of premenopausal women in the Harvard Study of Moods and Cycles showed that those who reported prenatal Discussion DES exposure had lower estradiol concentrations than those who did not report DES exposure (18). In the absence of In this study, we found evidence that women who were medical record confirmation, DES exposure status is likely to prenatally exposed to DES compared with those who were not be misclassified, although the magnitude of the effect of this on had less metabolism of parent estrogens in the 2 pathway; this previous study findings is unknown. In our study, which association remained after adjustment for total estrogens and includes confirmation of DES exposure status for all partici- adjustment for some other factors that may influence estrogen pants, results suggest that parent estrogen concentrations, metabolism, such as BMI, parity, years since menopause, or including estradiol, are somewhat greater in DES-exposed post- recent alcohol use. In the early 1970s, young women who had menopausal women. been exposed prenatally to DES demonstrated a strikingly high This study was not a random sample of the entire combined relative risk of vaginal cancer (1). With long-term follow-up, cohort study. The women who were included were likely exposed multiple other adverse health outcomes were linked to this to a high cumulative dose of DES in utero, and the majority had exposure, including precancerous lesions of the cervix (3), and VEC, which is associated with high DES dose and early exposure in a modest increase in breast cancer (2). These findings stimu- pregnancy (10). It was also limited in sample size. Nonetheless, it lated extensive laboratory investigations pursuing possible demonstrated a pattern of results consistent with a relative underlying biologic mechanisms. decrease in levels of 2-hydroxylation pathway metabolites Some evidence in mice suggest that prenatal DES exposure and in the ratio of these to total parent estrogens and to 16- may affect hormone concentrations later in life. An in vitro hydroxylation pathway metabolites in the prenatally DES study of total estrogen, testosterone, and progesterone in exposed compared with unexposed women. This profile has been mouse ovarian tissue showed significantly higher secretion associated with an increased risk of breast cancer in postmeno- in the DES-exposed animals (5). These hormonal changes may pausal women (6). Although the association between DES and be caused by epigenetic alterations. Recent data in mice (15) estrogen metabolism is in the direction of higher breast cancer risk show that maternal exposure during pregnancy to environmen- for the prenatally DES exposed and could therefore possibly tally relevant doses of BPA (chemically similar to DES) induces explain some of their modestly increased risk compared with the sex-specific, dose-dependent (linear and curvilinear), and brain unexposed, it will take a larger study to quantify the magnitude of region–specific changes in expression of genes encoding the effect (2). Because the number of participants studied was estrogen receptors and estrogen-related receptor-g in juvenile small, we tried to account only for the major influences on offspring. In the same study (15), BPA altered mRNA levels estrogen metabolism. We did not collect information on dietary
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Figure 1. Percent change (95% CI) in parent estrogens, estrogen metabolites, and ratios for DES exposed compared with unexposed women with adjustment for age, years since menopause, BMI, parity, and recent alcohol intake.
intake or physical activity, and we think it unlikely that there endocrine disruption in humans during the prenatal develop- would be a strong association between these factors and whether mental period. In this study, wehadtheuniqueopportunityto the participant's mother took DES during the daughter's preg- assess hormone concentrations in women with documented nancy. Our hypothesis was that prenatal exposure to DES effects exposure to an established endocrine disrupting chemical estrogen metabolism and that alterations in estrogen metabolism in utero. Our preliminary findingssuggestthatprenatalexpo- persist after menopause. Because we believe the effects of DES on sure to endocrine disruptors may influence estrogen metabo- estrogen metabolism would occur prenatally and therefore pre- lism many years later. cede the adverse reproductive events we have chronicled (9), we did not adjust for those intervening events in the analyses of DES Disclosure of Potential Conflicts of Interest exposure and estrogen metabolism. E.E. Hatch is a consultant/advisory board member for UpToDate (author This area of research has great potential to improve our on DES). No potential conflicts of interest were disclosed by the other understanding of the biological mechanisms associated with authors.
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Authors' Contributions Acknowledgments Conception and design: R. Troisi, E.E. Hatch, J.R. Palmer, L. Titus, R.N. Hoover The work was funded through contracts with the Division of Cancer Development of methodology: R. Troisi, L. Titus Epidemiology and Genetics, NCI. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): E.E. Hatch, J.R. Palmer, L. Titus, X. Xu, R.N. Hoover The costs of publication of this article were defrayed in part by the Analysis and interpretation of data (e.g., statistical analysis, biostatistics, payment of page charges. This article must therefore be hereby marked computational analysis): R. Troisi, J.N. Sampson, R.N. Hoover advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Writing, review, and/or revision of the manuscript: R. Troisi, E.E. Hatch, this fact. J.R. Palmer, L. Titus, X. Xu, R.N. Hoover Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R. Troisi Received January 29, 2018; revised May 2, 2018; accepted July 20, 2018; Study supervision: E.E. Hatch, L. Titus published first July 26, 2018.
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Estrogen Metabolism in Postmenopausal Women Exposed In Utero to Diethylstilbestrol
Rebecca Troisi, Elizabeth E. Hatch, Julie R. Palmer, et al.
Cancer Epidemiol Biomarkers Prev 2018;27:1208-1213. Published OnlineFirst July 26, 2018.
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