대한내분비외과학회지：제1권 제2호 □ 원 저 □ Vol. 1, No. 2, October, 2001

Effect of Endogenous on Human Papillary Thyroid Cancer: Alteration of Urinary Profile in Pre- and Post-operative Cases

Woung Youn Chung, Jin Hak Suh, Bong Chul Chung1 and Cheong Soo Park

Department of Surgery, Yonsei University College of Medicine, Bioanalysis & Biotransformation Research Center, 1Korea Institute of Science and Technology, Seoul, Korea

유두상 갑상선암 발생에 대한 내인성 스 Key Words: Endogenous , , , Papil- 테로이드의 영향 lary thyroid cancer 중심 단어: 내인성 스테로이드, 안드로겐, 에스트로겐,

1 유두상 갑상선암 정웅윤․서진학․정봉철 ․박정수 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ 연세대학교 의과대학 외과학교실, 1한국과학기술연구 목적: 스테로이드 호르몬은 다양한 종양의 성장에 기여하는 것은 잘 알려져 있다. 여러 보고에서 외인성 안드로겐이 갑상 원 도핑콘트롤센터 선 호르몬과 갑상선 세포의 성장에 기여한다는 사실이 증명 되었으나, 갑상선 암에서의 안드로겐의 역할은 아직 분명히 밝혀지지 않았으며, 최근의 연구에서는 갑상선 조직에서 에 스트로겐 수용체의 존재에 관하여, 갑상선암의 에스트로겐 의존성의 가능성을 제안해왔으나, 에스트로겐 수용체 양성율 INTRODUCTION 과 종양의 생물학적 양상과의 관계에 대한 명확한 결론을 얻 지는 못하였다. 이에 연구자들은 갑상선 암에서의 안드로겐 Steroid hormones are known to influence the development 과 에스트로겐의 역할을 규명하고자 본 연구를 시행하였다. 대상 및 방법: 유두상 갑상선암 환자의 수술전과 수술후의 of a number of different tumors. Clinical observations also 소변 견본과 정상여성의 소변 견본에서 안드로겐, 코티코이 support that the biological behavior of differentiated thyroid 드(corticoid), 에스트로겐 호르몬 프로파일의 변화를 gas cancer is affected by steroid hormones, such as . For chromatography/mass spectrometry/selected ion-monitoring (GC/ this reason, the incidence and distribution of cytosolic and/or MS/SIM) system를 통해 분석하였다. 또한, 호르몬의 불균형 이나 산화대사가 갑상선 암과의 연관성이 있는지 간접적으 nuclear androgen receptors (AR) have been analyzed in both 로 측정하기 위하여 관계 있는 호르몬 농도비를 측정하였다. normal and pathological human thyroids. (1-3) Several studies 결과: 수술전 측정된 11-DOKS/17-OHCS비가 17-OHCS 양 (4-6) have demonstrated the effects of exogenous androgens 의 감소로 인하여 의미 있게 증가하였다. Catechol 과 2-OH E1을 포함한 에스트로젠의 측정량이 다른 에스트로겐의 대 (, , etc.) on thyroid hormone and 사산물에 큰 변화 없이 수술전 유두상 갑상선암 환자에서 유 thyroid cell growth. However, their results are conflicting, and 의하게 증가하였다. 수술전 16-OH E1/2-OH E1 비는 수술후 the role of androgens on thyroid cancer has not been clearly 측정치에 비해 통계적으로 유의하게 낮은 결과를 보였다. 결론: 이상의 결과로 부신피질 홀몬 결핍에 의한 안드로겐의 elucidated. 체내 변화는 유두상 갑상선암의 발생에 영향을 줄 것으로 생 Various experimental results suggest that the growth regu- 각되며, 또한 에스트로겐 대사과정 중에 2-hydroxylation 과정 lation for thyroid cancer, as well as for the normal thyroid 의 증가는 유두상 갑상선암과 연관성이 있을 것으로 사료된 gland, appears to depend upon the thyroid stimulating hormone 다. (Korean J Endocrine Surg 2001;1:259-266) (TSH). Therefore, suppression therapy of TSH with thyroid hormone is considered to be effective against differentiated 책임저자：박정수, 서울시 서대문구 신촌동 134 thyroid carcinoma. Thyroid hormone, arising endogenously or ꂕ 120-752, 연세대학교 의과대학 외과학교실 exogenously, can affect the oxidations of several steroids, Tel: 82-2-361-5540, Fax: 82-2-313-8289 including (E2) and , which are sensitive to E-mail: [email protected] changes in thyroid function. (7,8) With regard to estradiol, the 게재승인일：2001년 10월 23일 논문의 요지는 2000년 제 7 차 아시아 내분비외과학회 학술대회 extent of hydroxlyation at C-2 or C-16α position has been 에서 발표되었음. found to correlate with the risk for estrogen-dependent disease, 259 260 대한내분비외과학회지：제1권 제2호 2001 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ such as breast cancer and endometrial cancer of the uterus. control pills or estrogen. (9,10) Recent studies dealing with the presence of estrogen Early morning urine samples were obtained. The collected receptors (ER) on thyroid tissue suggested the possibility that urine samples were stored at -20oC until analysis. Creatinine thyroid carcinoma is dependent on , but no definite was measured by a Jaff method. conclusions have been drawn between the biological behavior 3) Gas chromatography-mass spectrometry of the tumors and the ER-positivity. The present study was designed to evaluate the role of The Hewlett-Packard GC-MS system consisted of a Model androgens and estrogens in human thyroid cancer. The alte- 5890A gas chromatography, a Model 5970B mass-selective ration of androgen, corticoid, and estrogen profiles were detector, and a HP 59970C MS chemstation. The GC column investigated in the urine of pre- and post-operative patients with used for androgen analysis was a 17 m×0.2 mm (internal thyroid cancer and normal female subjects by the gas chromato- diameter) fused silica capillary, coated with methyl siloxane graphy/mass spectrometry/selected ion-monitoring (GC/MS/ (film thickness: 0.11μm). For estrogen analysis, a fused- silica SIM) system. Also, to indirectly determine whether a hormonal capillary coated with crosslinked 5% phenylmethyl siloxane (25 imbalance and/or oxidative metabolism would be associated m×0.2 mm×0.33μm) was used. The carrier gas (helium) flow with thyroid cancer, the related hormonal concentration ratios rate was 0.85 ml/min, and the split ratio was 1：13. The GC were estimated. temperature program was as follows: in the case of androgens, the initial temperature (180oC) was programmed at 4oC/min to MATERIALS AND METHODS 300oC and maintained for 2 min. And for estrogens, the initial temperature of 180oC was increased to 260oC at a rate of 20oC/ 1) Chemicals min and held there for 6 min. Then, it was increased to 275oC Androgen, corticoid, and estrogen standards were purchased at a rate of 2oC/min and held for 8 min. Finally, it was from Sigma Chemical Co., St. Louis, MO 63178, U.S.A. increased to 300oC at a rate of 15oC/min and held for 10 min. o d2-Estradiol used as an internal standard for the estrogen profile The injector temperature was 300 C, the transfer line was was purchased from MSD Isotope (Montreal, Canada). Serdolit 300oC, and the ion source was 200oC. The mass spectrometer AD-2 resin (particle size: 0.1∼0.2 mm) was purchased from was operated at 70 eV in the electron-impact (EI) mode. The Serva (D-69115 Heidelberg. Carl-Benz-Str.7, Germany). β- selected ion-monitoring mode was used for quantifying the 21 Glucuronidase/arylsulfatase from Helix Pomatia was purchased androgen and 20 estrogen metabolites. The dwell time for each from Boehringer Mannheim (Germany): β-Glucuronidase acti- ion was set at 50 msec. vity was 5.5 U/mL (at 39oC) and arylsulfatase activity was 2.6 4) Extraction of androgens and corticoids U/mL (at 38oC). Silylating reagents, N-methyl-N-trimethylsilyl- heptafluorobutyramide (MSHFB) was purchased from Ma- A preconditioned Serdolit AD-2 resin was poured into a cherey-Nagel (D-5160 Dren, Germany). N-methyl-N-trimethyl- Pasteur pipette (0.5 cm ID) to 3 cm. Three ml of urine and silyl-trifluoroacetamide (MSTFA), trimethylsilylchloride (TMCS) an internal standard (cholesteryl isobutyrate, 0.2μg) were and N-trimethylsilyl-imidazole (TMSIm) were purchased from applied to the column. After the column was washed with 3 Sigma (St. Louis, MO, U.S.A). Ethylacetate and ether were of ml of water, the androgens were eluted three times with 1 ml a high purity “HPLC solvent” grade. Ether and deionized water of methanol. The eluant was evaporated to dryness in a rotary were distilled before use. evaporator. To carry out enzyme hydrolysis, the residue was then dissolved in 1 ml of acetate buffer (0.2 N, pH 5.0) 2) Subjects and sample-collection containing 50μl of β-glucuronidase/arylsulfatase (from Helix Urine samples were obtained from 54 female patients (49.4 Pomatia). The sample was incubated overnight at 37oC, or for ±12.6 y) who were diagnosed with thyroid papillary cancer. 3 hrs at 55oC. After the hydrolysis, 100 mg of potassium No patients were treated with exogenous hormones and all carbonate was added, and the pH was adjusted to 9.0. The subjects showed normal thyroid function (T3; 100.93±16.31 mixture was extracted with 5 ml of diethylether, and the ng/dl, T4; 11.27±2.01μg/dl, TSH; 0.77±0.40μU/ml, Mean± organic layer was transferred to another tube for vacuum SD). As a normal control, the urine of 20 women (48.3±11.9 evaporation. The residue was dried in a vacuum desiccator over y) with no evidence of benign or malignant thyroid disease P2O5-KOH. The residue was dissolved in 50μl of the reagent were included in this study. No women were taking birth mixture (MSHFB/TMCS/TMSIm, 2：2：1 volume ratio) and Chung WY, et al：Effect of Endogenous Steroids on Human Papillary Thyroid Cancer 261 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ heated for 10 min at 60oC. After heating, 2μl aliquots were TMCS, 100：1 volume ratio) at 60oC for 30 min. Two μl injected into the GC column by an autosampler. aliquots were injected into the GC column by an autosampler.

5) Extraction of estrogens 6) Assay

The urine sample (3 ml) and internal standard (d2-estradiol; The following 21 androgens and corticoids: 0.5μg) were applied to the column of preconditioned Serdolit (An), (Et), (DHEA), AD-2 resin. After washing with water, the free and conjugated 4- (Δ4-Dione), 5- (Δ5-Diol), estrogens were eluted with methanol, and the eluant was eva- testosterone (Te), dihydrotestosterone (DHT), 16 α-hydroxy porated to dryness. Enzyme hydrolysis was performed using β- DHEA (16α-OH DHEA), 5-androstene-3α, 16β, 17β-triol glucuronidase/arylsulfatase (from Helix Pomatia) with acetate (Δ5-AT), tetrahydrodeoxycorticosterone (THDOC), tetrahydro- buffer (0.2 N, pH 5.0) and ascorbic acid (1 mg/ml) at 37oC 11-deoxycortisol (THS), tetrahydro-11-dehydrocorticosterone (overnight) or 55oC (3 hrs). After the hydrolysis, potassium (THA), (THE), 5β-tetrahydrocortisol (THF), carbonate was added, and the mixture was extracted with 5α-tetrahydro-cortisol (5α-THF), α-cortolone, 5β-tetrahydro- ethylacetate. Then the organic layer was evaporated until dry. cortico-sterone (THB), β-cortolone, β-cortol, α-cortol, 5α- The residue was derivatized by the reagent mixture (MSTFA/ (5α-THB), and 14 estrogens:

Table 1. Concentration of urinary androgens and corticoids in normal female subjects and pre- and post-operative patients with thyroid cancer (μmol/g of creatinine) ꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚ Patients with thyroid cancer Normal female (n=20) ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Endogenous androgens & corticoids Pre-operative (n=29) Post-operative (n=25) P-value ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Mean Range Mean Range Mean Range ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ 11-Deoxy-17-ketosteroids and their metabolites (11-DOKS) Androsterone (An) 7.29 3.79∼13.51 7.02 2.70∼15.23 7.15 2.34∼13.35 NS* Etiocholanolone (Et) 6.63 2.24∼12.30 6.09 2.10∼12.13 7.14 2.16∼12.91 NS Dehydroepiandrosterone (DHEA) 1.16 0.28∼4.35 0.99 0.12∼3.84 1.04 0.09∼4.17 NS 4-Androstenedione (Δ4-Dione) 0.80 0.11∼2.17 0.80 0.12∼2.16 0.74 0.15∼1.79 NS 5-Androstenediol (Δ5-Diol) 0.51 0.14∼1.58 0.39 0.12∼0.76 0.49 0.10∼1.21 NS Testosterone (Te) 0.37 0.09∼0.84 0.35 0.10∼0.85 0.35 0.11∼0.72 NS Dihydrotestosterone (DHT) 0.72 0.16∼1.99 0.74 0.13∼1.93 0.70 0.12∼1.62 NS 16α-Hydroxy DHEA (16α-OH DHEA) 3.17 0.85∼8.08 3.12 0.87∼7.22 3.05 1.03∼7.98 NS 5-Androstene-3α,16β,17β-triol (Δ5-AT) 2.01 0.19∼5.44 1.90 0.31∼5.18 1.85 0.22∼5.78 NS

17-Hydroxycorticosteroids and their metabolite (17-OHCS) Tetrahydrodeoxycorticosterone (THDOC) 0.23 0.03∼0.77 0.07 0.02∼0.22 0.21 0.03∼0.62 ＜0.05 Tetrahydro-11-deoxycortisol (THS) 0.82 0.14∼1.93 0.20 0.05∼0.50 1.00 0.15∼2.18 ＜0.05 Tetrahydro-11-dehydrocorticosterone (THA) 0.60 0.07∼1.71 0.20 0.04∼0.41 0.69 0.16∼1.86 ＜0.05 Tetrahydrocortisone (THE) 13.40 2.73∼29.29 7.04 1.35∼17.43 12.94 3.65∼33.54 ＜0.05 5β-Tetrahydrocortisol (THF) 9.10 3.15∼24.08 2.69 0.41∼7.59 9.36 2.46∼25.15 ＜0.05 5α-Tetrahydrocortisol (5α-THF) 6.08 1.42∼17.10 1.24 0.18∼4.95 6.84 1.22∼23.72 ＜0.05 α-Cortolone 6.03 1.41∼13.24 2.03 0.30∼4.61 5.65 1.44∼12.64 ＜0.05 5β-Tetrahydrocorticosterone (THB) 2.31 0.33∼6.19 0.31 0.05∼1.07 2.44 0.26∼7.39 ＜0.05 β-Cortolone 2.64 0.49∼6.60 1.05 0.21∼2.54 2.93 0.41∼7.12 ＜0.05 β-Cortol 1.62 0.19∼5.71 0.53 0.11∼1.43 1.92 0.19∼7.07 ＜0.05 α-Cortol 2.07 0.58∼6.63 0.56 0.25∼1.41 2.36 0.47∼8.69 ＜0.05 5α-Tetrahydrocorticosterone (5α-THB) 1.71 0.22∼5.43 0.25 0.02∼0.70 1.77 0.11∼6.16 ＜0.05 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ *NS = not significant. 262 대한내분비외과학회지：제1권 제2호 2001 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ (E1), 17β-estradiol (E2), (E3), 16α-hydroxyestrone (16α- distributed, and the statistical significance of the difference in OH E1), 16-ketoestradiol (16-Keto E2), 17- (17-epi these variables among the three groups (pre- and post-operative E3), 16-epiestriol (16-epi E3), 6-ketoestriol (6-Keto E3), cancer patients and normal controls) were evaluated by one- 2-hydroxyestrone (2-OH E1), 2-hydroxyestradiol (2-OH E2), way analysis of variation (ANOVA) using statistical analysis 2-methoxyestrone (2-Meo E1), 2-methoxyestradiol (2-Meo E2), software (SAS, Cary, NC). Distributions of 11-DOKS/17- 2-methoxyestradiol-3-methylether (2,3-diMeo E2), 2-hydroxye- OHCS and 16α-OH E1/2-OH E1 also appear to be normal in stradiol-3-methylether (2-OH-3-Meo E2) were measured. All each three groups. With regard to the ratios of pre- and values were corrected for concentration of urinary creatinine. post-operative cases, statistical analysis for the significance of All urine samples were analyzed in separate batches for the the difference from the normal value was conducted by the two groups within a 1-month period together, with one duplicate t-test for two independent means or, when appropriate, by the quality-control sample for each batch. The quality-control sam- paired t-test. The Pearson correlation coefficient was used to ples used were pooled urine samples from normal individuals. analyze the univariate relationship between11-DOKS/17-OHCS The recovery range of the androgen and corticoid extraction and TSH value. As the value of significance, P＜0.05 was method was 72.33∼94.54%. It was found to be reproducible accepted. and quantitative. The Cvs of intraday analysis was 1.43∼ 10.86%, and that of interday analysis was 0.96∼9.98%.(11) In RESULTS regard to the extraction method for estrogens, the recovery 1) Urinary profile of androgens and corticoids range was 80.97∼97.81%, inter- and intra-assy Cvs were 0.24 ∼10.52%, 1.05∼10.24%, respectively. This method also had The concentration of 21 androgens and corticoids in the urine good quality-control data.(12) of pre- and post-operative patients with thyroid cancer and normal female controls were determined using the GC/MS/SIM 7) Statistical analysis method and the ranges and mean values are presented in Table All directly measured hormone variables were normally 1. The urinary levels of the majority of adrenal androgen

Table 2. Concentration of urinary estrogens in normal female subjects and pre- and post-operative patients with thyroid cancer (nmol/g of creatinine) ꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚ Patients with thyroid cancer Normal female (n=20) ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Endogenous androgens & corticoids Pre-operative (n=29) Post-operative (n=25) P-value ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Mean Range Mean Range Mean Range ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Major estrogens & 16α-Hydroxylation metabolites Estrone (E1) 16.24 5.12∼34.01 14.20 4.60∼32.49 14.34 5.62∼28.43 NS* 17β-Estradiol (E2) 14.50 7.13∼27.63 12.81 4.71∼25.02 12.79 5.46∼26.23 NS Estriol (E3) 19.03 6.48∼43.12 18.33 6.52∼43.28 18.16 6.89∼42.27 NS 16α-Hydroxyestrone (16-OH E1) 17.20 5.63∼37.42 18.65 5.28∼35.50 19.38 6.95∼33.91 NS 16-Keto E2 8.67 2.82∼17.94 8.36 2.29∼16.84 8.53 2.95∼15.42 NS 17-epi E3 9.51 3.68∼25.43 11.11 4.01∼24.76 10.80 3.90∼21.28 NS 16-epi E3 12.02 4.66∼22.44 12.93 5.11∼24.15 12.27 4.70∼20.45 NS 6-Keto E3 10.66 3.27∼21.33 10.92 3.40∼19.67 10.87 2.97∼23.72 NS 2-Hydroxylation metabolites (Catechol estrogens) 2-OH E1 120.7 34.38∼281.7 538.3 154.7∼1324.6 222.2 112.4∼401.8 ＜0.05 2-OH E2 15.63 5.23∼25.64 47.03 8.97∼98.65 20.11 6.72∼39.24 ＜0.05 2-Methoxy E1 (2-Meo E1) 36.40 10.87∼69.24 98.03 26.31∼265.0 44.28 12.81∼91.48 ＜0.05 2-Meo E2 19.71 7.17∼44.39 98.98 25.02∼232.5 26.18 9.51∼52.31 ＜0.05 2,3-diMeo E2 25.43 7.49∼56.98 58.97 15.43∼112.4 30.87 8.67∼72.17 ＜0.05 2-OH, 3-Meo E2 20.84 6.40∼49.83 62.03 19.91∼134.2 28.58 7.78∼65.63 ＜0.05 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ *NS = not significant. Chung WY, et al：Effect of Endogenous Steroids on Human Papillary Thyroid Cancer 263 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ

Fig. 1. Distributions of the urinary level of 2-OH E1 (A) and concentration ratio of 16α-OH E1/2-OH E1, (B) in normal female subjects and pre- and post-operative patients with thyroid cancer.

metabolites (11-deoxy-17-ketosteroids and their metabolites) were not different significantly in the three groups, and their P values were evaluated to be ‘not significant (NS)' from the one-way ANOVA test. On the contrary, the excretion of 17- hydroxycorticosteroids and their metabolites were significantly lower in pre-operative patients with thyroid cancer than those measured in normal female subjects and post-operative patients.

2) Urinary profile of estrogens

The urinary levels of 14 estrogen metabolites were also estimated quantitatively, and these results (mean and range) are described in Table 2. From Table 2, it can be seen that Fig. 2. Distribution of the ratio of 11-DOKS/17-OHCS in normal pre-operative patients with thyroid cancer have a significantly female subjects and pre- and post-operative patients with thyroid cancer. elevated excretion of catechol estrogens (2-OH E1, 2-OH E2, 2-Meo E1, 2-Meo E2, 2,3-diMeo E2 and 2-OH-3-Meo E2). Measurements of urinary estrogen metabolites also showed that the concentrations of catechol estrogens were decreased to the 3) Relative ratios of 11-DOKS/17-OHCS and 16α-OH values slightly higher than those of normal subjects after E1/2-OH E1 operation. The change of the 2-OH E1 level in the three groups was illustrated in Fig. 1A. With the exception of catechol The ratio of the sum of 11-deoxy-17-ketosteroids and their estrogens, there were no significant alterations in major metabolites to the sum of 17-hydroxycorticosteroids and their estrogens (E1, E2 and E3) and 16α-hydroxylation metabolites metabolites (11-DOKS/17-OHCS) was determined in pre- and (16α-OH E1, 16-Keto E2, 17-epi E3, 16-epi E3 and 6-Keto post-operative patients with thyroid cancer and the corres- E3) between the normal group and the cases. ponding control group (Table 3). The highest mean value of the 11-DOKS/17-OHCS ratio was observed in pre-operative pa- tients. This ratio was markedly decreased in post-operative 264 대한내분비외과학회지：제1권 제2호 2001 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ

Table 3. Relative ratios of 11-DOKS/17-OHCS and 16α-OH E1/ chanism for the growth-modulation of thyroid cancer involves 2-OH E1 in normal female subjects and pre- and post- an action of androgens directly on thyroid tissue via specific operative patients with thyroid cancer cellular receptors for androgens. (1-3) Prinz et al. (1) demon- ꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚ Patients with thyroid cancer strated that cytosolic androgen receptors are present in all types Normal ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ of thyroid lesions, and the receptor content is higher in dif- female (n=20) Pre-operative Post-operative ferentiated thyroid cancer when compared to nonmalignant (n=29) (n=25) lesions. ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ 11-DOKS/17-OHCS In this investigation, to assess the influence of androgen- Mean 0.52 1.52 0.53 dominant milieu observed in the cancer cases on TSH, the Range 0.17∼1.15 0.63∼4.71 0.22∼1.26 linear-correlation analysis between the ratio of 11-DOKS/17- SD 0.20 0.78 0.24 OHCS and TSH value was performed. 11-DOKS (11-deoxy-17- P-value ＜0.05 NS* ketosteroids and their metabolites) are originated in adrenal 16α-OH E1/2-OH E1 androgens and 17-OHCS (17-hydroxycorticosteroids and their Mean 0.18 0.05 0.16 metabolites) are corticoid metabolites. As a result, no signi- Range 0.05∼0.35 0.01∼0.12 0.05∼0.38 SD 0.08 0.03 0.10 ficant relationship was obtained in normal subjects and patients P-value ＜0.01 NS with thyroid cancer (data not shown). Evidence from the ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ present study supports the hypothesis that thyroid tissue, par- *NS: not significant. ticularly neoplastic tissue, may possess the potential for direct growth-regulation by androgens. And, it is possible that andro- gens may exert such regulation effect via a specific imbalance patients and normal controls. Fig. 2 represented the distribution of androgens and corticoids in human thyroid cancer. of 11-DOKS/17-OHCS values in the three groups. Thyroid dysfunction in humans is known to alter the excre- The ratio of 16α-OH E1/2-OH E1 was determined, and the tory pattern of estrogen metabolites, suggesting that thyroid range and mean value was compared between the cases and the hormone directly influences the oxidative metabolism of estra- normal groups (Table 3). In pre-operative patients with thyroid diol. (8) So, we performed a profile analysis for 16 estrogen cancer, the lowest range and mean value of 16α-OH E1/2-OH metabolites in the urine of normal female subjects and pre- and E1 were observed (P ＜ 0.01). This changed ratio was returned post-operative patients with thyroid cancer using the GC/MS/ to the normal value in the corresponding post-operative pa- SIM system. Also, to evaluate the extent of estradiol hydroxy- tients. The distribution of the ratio of 16α-OH E1/2-OH E1 lation at C-2 and C-16α position which are the two primary is represented in Fig. 1B. and competing sites of estrogen-oxidation, the ratio of 16α- hydroxyestrone to 2-hydroxyestrone (16α-OH E1/2-OH E1) DISCUSSION was compared with the cases and the corresponding control group. The present study demonstrates that the oxidative meta- It is generally accepted that androgens may play a physio- bolism of estrogens is altered by elevating C-2 hydroxylation logical role on the thyroid. The previous data suggested that without a significant change of 16α-hydroxylation in pre- physiologic androgenic milieu may influence the growth of operative patients with thyroid cancer. The increased 2-OH E1 thyroid tumors. (1) Our results from the urinary profile of an- is further metabolized to produce the other catechol estrogens drogens and corticoids (Table 1) show that an androgen- (2-Meo E1, 4-Meo E2, 2,3-diMeo E2, 2-OH-3-Meo E2 and dominant environment caused by a significant decrease of 2-Meo E2), whose levels are markedly higher than normal corticoids is associated with thyroid cancer. values (Table 2). In post-operative patients with thyroid cancer, However, the effect of androgens on the induction and/or the urinary levels of catechol estrogen, including 2-OH E1, are progression of thyroid cancer has been explained through a decreased and distributed between those of normal subjects and direct or indirect mechanism. In the studies of Hofmann et al., pre-operative cases (Fig. 1A). On the other hand, major (4) it has been indicated that androgens may indirectly modu- estrogen (E1, E2 and E3) and their C-16α metabolites (16α- late growth of radiation-induced thyroid tumors by increasing OH E1, 16-epi E3 and 17-epi E3) were not significantly altered TSH production, which may enhance thyroid cancer develop- in the three groups (P＞0.05). ment by growth-promoting actions. (13-15) An alternative me- The ratio of 16α-OH E1/2-OH E1, an index of the relative Chung WY, et al：Effect of Endogenous Steroids on Human Papillary Thyroid Cancer 265 ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ strengths of the two competing oxidative pathways, was E1 was decreased in pre-operative cancer patients. However, significantly lower in pre-operative patients with thyroid cancer further researches will be necessary to determine whether such than those found in post-operative patients and in normal metabolic alterations, in fact, influence the risk for thyroid female subjects. This decrease was mostly responsible for cancer, or merely accompany the change in risk. raising urinary 2-OH E1 excretion. The variation of 16α-OH E1/2-OH E1 ratio in the three groups was represented in Fig. REFERENCES 1b. As shown in Fig. 1b, in post-operative patients with thyroid cancer, the relative ratio showed no significant difference from 1) Prinz RA, Sandberg L, Chaudhuri PK. Androgen receptors in the normal value, not in accordance with the distribution in human thyroid tissue. Surgery 1984;96:996-1000. absolute amounts of 2-OH E1. 2) Sheridan PJ, Aufdemorte TB, Triplett RG, Holt G, Martin PM. 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