[CANCER RESEARCH 28, 328-337, February Ig68] Selective Retention of Estrogen Isomers in Estrogen-dependent Breast Tumors of Rats Demonstrated by in Vitro Methods Lars Terenius Departmen~ of Pharmacology, University of Uppsala, Uppsala, Sweden SUMMARY trogen-responsive DMBA tumors to be 8-20 times the muscle concentration around 100 minutes after subcutaneous injection Huggins' rat mammary tumors were induced by treatment in saline. She also found a lower accumulation in estrogen-un- with 7,12-dimethylbenz (a) anthracene (DMBA) and the estro- responsive DMBA tumors, indicating that estrogen uptake and gen-dependent ones were selected. The tumors avidly took up retention is characteristic for the estrogen dependence of the potent estrogens 17fl-estradiol and meso-hexestrol in vivo, DMBA-induced tumors. but they did not take up the much less estrogenic racemic This communication describes estrogen uptake and wash-out hexestrol. The potent estrogens in the tumor were not easily experiments with the DMBA-induced tumor. The character- washed out in vitro. Diaphragm neither took up nor retained istics and specificity of these processes is studied. Most ex- potent estrogens significantly. periments have been carried out in vitro, which permits the The selectivity in vitro was most clearly visible as a selective use of slices from the same tumor in experiments under different retention of the potent estrogens in the tumor. When tumor conditions. By this means one can relate all effects to appro- slices and diaphragm strips were incubated with labeled 17fl- priate controls and the problem of variation between tumors estradiol, the tumor/diaphragm concentration ratios became is eliminated. Methods for analysis of estrogen uptake in vitro about 2; but after a subsequent wash-out, in the presence of have been developed for the mouse uterus (22, 25). Similar a large excess unlabeled 17fl-estradiol, the concentration ratios methods have been used for the tumor. Strips of diaphragm increased to about 4. meso-Hexestrol, but not the less estrogenic from the tumor-bearing rat are included in all experiments, racemic hexestrol, was selectively retained by the tumor in the the diaphragm being regarded as a non-target, control tissue. same way. Unlabeled 17a-estradiol, 17fl-estradiol, the synthetic After this work was completed a communication by Jenscn estrogens meso-hexestrol, racemic hexestrol, or the demethylated et ol. (13) appeared, also dealing with the uptake in vitro of analog of the estrogenic acid, methallenestril, suppressed the 17fl-estradiol by the DMBA-induced rat mammary tumor. In retention of labeled 17fl-estradiol by the tumor but not by the many respects, the observations made by these authors agree diaphragm when present during uptake. These estrogenic with the present ones, even if the experimental conditions stereoisomers were inhibitory to an extent which was correlated differ. with their estrogenic potencies. The degree of uptake and re- tention of 17fl-estradiol in the tumor was diminished by a MATERIALS AND METHODS sulfhydryl inhibitor, N-ethylmaleimide, present during uptake or wash-out. The uptake of 17fl-estradiol by adipose tissue was Radioactive Compounds and Nonradioactive Estrogen high, but showed no specificity. Preparations. Tritium-labeled 17fl-estradiol (labeled in the 6, In all these respects the selectivity of estrogen uptake and 7 positions) was obtained from New England Nuclear Corp. retention in the DMBA tumor is similar to that of the mouse Its specific activity was 132 ~c//~g. Tritium-labeled isomers uterus. This similarity indicates the presence of specific es- of hexestrol were prepared by the author as described else- trogen receptors in the tumor and favors the view that estro- where (21). Their specific activities were 150 ~c/~g. The gens exert a direct influence on the growth of this tumor. radiochemical purity of the radioactive compounds was con- trolled at intervals by thin-layer chromatography on silica gel INTRODUCTION with chloroform:acetic acid (85:15, v/v) or chloroform:di- ethylamine (80:20, v/v) as developer. At times of use the Estrogen-responsive tissues, such as the mammalian uterus radiochemical purity was about 98%. and vagina, take up and retain estrogens (e.g., 8, 14, 20). Most 2 Abbreviations : DMBA, 7,12-dimethylbenz(a) anthracene ; 17fl- rat mammary tumors induced by treatment with DMBA 2 also estradiol, estra-l,3,5(10)triene-3,17fl-diol; hexestrol, 3-4-bis(p-hy- are estrogen-responsive (6, 9). It has been found that this droxyphenyl)-n-hexane; methallenestril, dl-3-(6-methoxy-2-naph- tumor retains systemically administered 17fl-estradiol (15). thyl)-2,2-dimethylpentanoic acid; KRP, Krebs-Ringer phosphate Mobbs (17) found the concentration of 17fl-estradiol in es- buffer; KRPA, Krebs-Ringcr phosphate buffer with 2% albumin; NEM, N-ethylmaleimide; U-11100A, 1-(2-[p-(3,4-dihydro-6-meth- 1 Supported by the Swedish Cancer Society. oxy-2-phenyl- 1,1-naphthyl) phenoxy]-ethyl) pyrrolidine hydro- Received May 25, 1967; accepted October 15, 1967. chloride. 328 CANCER RESEARCH VOL. 28 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1968 American Association for Cancer Research. Estrogen Retention in Breast Tumors Nonradioactive 17fi-estradiol and the hexestrol isomers were Table 1 of the same preparation as previously used (21). Methallenes- Content of radioactivity a tril was a commercial specimen, m.p. 225-228~ (Vallestril | Rat Tumor Diaphragm Tumor G. D. Searle & Co., Chicago, Ill.), and the corresponding No. Treatment Medmm Medium I)iaphragra free phenol, dl-3- (6-hydroxy-2-naphthyl)-2,2-dimethylpentanoic A. Uptake a 3.6 -4- 0.13 e 1.85 __+ 0.09 1.95_+ 0.12 acid, m.p. 175-176~ was prepared by the author from 1 B. Wash-out 22~ b 2.2 ___ 0.13 0.42_+ 0.02 5.1 ___ 0.40 methallenestril by demethylation with hydrobromic acid. C. Wash-out 37~ c 1.53 ___ 0.10 0.29_ 0.01 5.5 _ 0.39 Animals and Assay Procedure. Albino rats of the Sprague- A. Uptake 3.8 _ 0.4 2.0 --4-_ 0.11 1.9 ___ 0.23 Dawley strain were supplied through the courtesy of Dr. 2 B. Wash-out 22~ 1.57 ___ 0.05 0.52_ 0.06 3.0 _ 0.35 Charles Huggins from his laboratory. Females were treated C. Wash-out 37~ 1.19 _ 0.06 0.26 _ 0.01 4.6 _ 0.29 with DMBA when they were 50, 53 and 56 days old. On these days 2 mg DMBA in 0.4 ml 15% fat emulsion (obtained from A. Uptake 5.2 __+ 0.4 2.6 ___ 0.11 2.0 -4- 0.18 3 B. Wash-out 22~ 2.5 -*- 0.12 0.94__+ 0.19 2.6 ___ 0.55 Dr. Huggins) was injected intravenously (10). About one C. Wash-out 37~ 1.68 _ 0.10 0.55_+ 0.03 3.1 _+ 0.19 month later the animals were inspected twice weekly and mam- mary tumors located by palpation. When a rapidly growing Uptake and effect of temperature on retention of 17fl-estradiol tumor appeared, the tumor-bearing animal was ovariectomized by the DMBA tumor and diaphragm in vitro. a Incubation for 1 hour at 37~ in 6 ml of KRPA (Krebs- (in most cases). When regression of the tumor was ascertained, Ringer phosphate buffer with 2% w/v albumin) and 0.003 #g 1.5 t~g of 17fl-estradiol-3-benzoate was injected subcutaneously 17fl-estradiol-3H (1.8 • 10 -9 M solution). in 0.15 ml olive oil every second day. If the regressed tumor b Preincubation as Group A; then incubation 1 hour at 22~ started to grow rapidly on the estrogen treatment, it was con- in other flasks with 6 ml of KRPA and 0.3 #g unlabeled 17fl- sidered to be estrogen-responsive and used for experimentation. estradiol (1.8 • 10 -7 M solution). Only a minor fraction (about 20%) of the animals developed c Preincubation as Group A; then incubation 1 hour at 37~ in tumors which were considered useful by these criteria. The other flasks with 6 ml of KRPA and 0.3 #g unlabeled 17fl- tumor was used for experimentation when its diameter was estradiol. 1-2 cm, 4 days after the last treatment with 17fl-estradiol-3- Concentration ratios tissue/medium calculated as benzoate. In cases where several tumors existed in one rat, only dpm/mg wet tissue "Medium" in this and all subsequent table dpm/#l medium " the most clearly estrogen-responsive one was utilized. headings refer to the medium in which the tissues were loaded After sacrifice of the animal the tumor was dissected free with radioactivity. from surrounding connective tissue. Tumor areas free from e Mean _ S.E. There were 6 tissue slices/group. necrosis and hemorrhage were sliced with a tissue slicer (type Stadie-Riggs). The thickness varied but always was ~1 mm. The first slice was discarded to exclude the outer capsule of Chemical identification of the radioactivity accumulated in the tumor. From the slices, strips weighing approximately 10 the tissues was performed as described earlier (21). mg were cut out. Simultaneously, diaphragm strips of the same weight were prepared. Two tumor strips and two diaphragm RESULTS strips were put into an incubating flask containing 6 ml of KRP pH 7.4, prepared from reagent chemicals and redistilled In the tables each animal has been treated separately since water (5). In most cases the KRP contained 2% (w/v) bovine the differences between tumors from different animals have albumin (Cohn fraction V, Sigma B grade). Aliquots of alco- been considered too great to permit averaging. holic stock solutions of radioactive as well as nonradioactive Ovariectomized Animals in Vitro.