Distribution of radioactivity in rat tissues after administration of tritiated 17α-ethynyl-19-norsteroids

Distribution of radioactivity in rat tissues after administration of tritiated 17α-ethynyl-19-norsteroids

97 DISTRIBUTIC~ GF RADIOACTIVITY IN RAT TISSUES AFTER ADMINISTRATIO~ OF TRITIATED 17a-ETHYNYL-19-NORSTEROIDS H. Watanabe, N.N. Saha ~-d D.S. layne F...

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DISTRIBUTIC~ GF RADIOACTIVITY IN RAT TISSUES AFTER ADMINISTRATIO~ OF TRITIATED 17a-ETHYNYL-19-NORSTEROIDS

H. Watanabe, N.N. Saha ~-d D.S. layne Food and Drug laboratories, Department o f National Health and Welfare,

Ottawa 3, Canada.

Received August 24, 1967 ABSTraCT Stably tritiated norethynodrel, noreth4~rone and ethynodlol diacetate were each administered to female rats in a dose of 0.i ~g per i00 g body weight. Radioactivity was assayed in uterus, ovaries, liver, kidneys, serum, adrenals, pituitary~ hypothalamus and cerebellum of --4-~is killed at intervals up to 4 hours after injection of the dose. No evidence was found of a selective uptake of the steroids by these tissues.

INTRODUCTI~ While there is no evidence that progesterone or other progestational steroids are selectively concentrated in tissues, Eisenfeld and Axelrod (i) have reported that relatively large doses of 17aethynyl-17~-hydroxTestr-5 (10)-en-3-one (noretkvnodrel) will partly inhibit the uptake of estradiol by uterine tissue. Their results suggested that this inhibition was competitive. Jensen and co-workers (2) have shown that 17a-ethyaylestradiol is selectively localized in uterine tissue, and the 3-cyelopentyl ether of this compound (quinestrQl) has been shown (3) to be stored ,inaltered in brain.

It seemed of

interest, therefore, to determine whether the 17a-ethynyl steroids comonly used as oral progestins were selectively concentrated in various rat tissues, and such a study was made possible by the

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availability of stabl~ tritiated norethyn~irel and the related compounds 17~-eth~n~yl-17~-hydroxyestr-4-en-3-one (norethi~irone) and 17a-ethynyl-3~, 17~,iiacetoxyestr-4-ene (ethynodiol diacetate).

MATERIALS AND ~THODS Norethindrone-6,7-3H, norethynodrel-6,7-3H and ethyaodiol diacetate-6,7-3H were prepared from estradiol-6,7-3H by Dr. K.I.H. Williams of the Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts. The specific activity was 11.6 ~c/~g based on norethlndrone. All the steroids were rigorously purified by th~n layer chromatography (4) immediately before use. Mature female Wistar rats weighing from 140 to 180 g were injected intravenously with 0.i ~g of tritiated steroid per I00 g body weight. Injections were made in 10% ethanolic saline. Animals were killed by e x s a ~ t i o n , at accurately timed intervals. Tissues were 4,~ediately excised and weighed. Accurately weighed se~aents (40-50 rag) of liver, kidney and uterus, were placed in separate liquid scintillation vials and 0.5 ml of hyamine hydroxide was added. After 16 hours the vials were heated at 60°0 for 3 hours. Ethanol (0.5 ml) and 15 ml of scintillation mixture containing 200 mg of dimethyl POPOP and 6 g of PPO per liter of toluene, were added to each vial. Radioactivity was determined in a Packard spectrometer and quench corrections were obtained by the use of 3H-toluene as internal stan~-~d. In the case of ovaries and various brain tissues, the entire tissue from each rat was used for the assay. Radioactivity in serum was determined by adding 0.3 ml of serum to 3 ml of hyamine hydroxide and 1.5 ml of ethanol, standing at room temperature for 16 hours, and counting in toluene scintillator as described above. RESULTS Following a single injection of 0.i ~g of 6,7-3H-estradiol per i00 g bod~ weight, the distribution of radioactivity in the tissues of I0 rats was essentially that reported by Jensen e_~ al (2). Radioactivity was concentrated in the uterus, and to a lesser extent in the pituitary.

This established that in our rats, under

our conditions, these well-known effects could be obtained. The results obtained after the injection of the same weight of 6,7-3H-norethindrone are shown in Figure I.

The data r@present the

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20,000-

p-

15,000-

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I0,000-¢9

o o ,000.

0 TiME

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I INJECTION

2 4 IN HOURS

Figure I. Distribution of radioactivity in rat tissues after injection of norethindrone-6,?-7-3H. The bars represent standard error of the means. Serum values are expressed as d.p.m, per 0.I ml. average of 6 experiments with a total of 36 rats. There was no evidence of accumulation of this steroid by any of the tissues studied, which included the ovaries as well as the tissues depicted in Figure i.

The relatively high levels of radioactivity present

in some of the tissues immediately after a~n~strstion of the dose decreased rapidly with time. When either 3H-norethynodrel or 3Hethynodiol diacetate were injected, the distribution pattern of radioactivity showed no discernible difference from that found with norethindrone and shown in Figure i. In a separate experLment involving five rate, the anterior pituitary, cerebellum and hypothalamus of each animal were separately assayed for radioactivity after the injection of 6,?-3H-norethindrone. No concentration of radioactivity above the serum levels was detected in any of these tissues.

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DISOUSSICW The organs most likely to be targets of contraceptive action by norethindrone, noretbynodrel and ethynodiol diacetate are the uterus and pituitary, and possibly also the ovary and hypothalamus.

The

results indicate that if any of these tissues accumulates radioactivity injected as these steroids, this accumulation is either very tr---itory, or is ~-detectable under the conditions of our experiments, which were adequate to detect the well-known localization of estradiol (2) in uterine tissue.

It is, of course, possible that a metabolite or

metabolites of the injected steroids might be produced and concentrated in very small amounts, but in interpreting these findings, it is important not to make the assumption that a hormone or other active agent is necessarily concentrated to s~y detectable extent in target tissue. Indeed, there is no evidence for such concentration with steroids other than estradiol and related estrogens. In view of these results it seems unlikely that noretbynodrel acts as a competitive inhibitor of estradiol uptake as suggested by Eisenfeld and Axelrod (i). These authors state that the level of aromatic contaminants in the noret~Tnodrel used in their studies was less than 0.05%. However, they administered 220 ~g of norethynodrel per i00 g of body weight to their rats, so that this dose ~

have contained up to 0.Ii pg per i00 g of body weight of

ethynylestradiol or other estrogen, an amount of the same order as that of their 3H--estradiol doses.

Jensen (2) has shown that

ethynylestradiol and mestranol administered at these levels are taken up by uterine tissue in a manner similar to that found with

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estradiol. The results of Eisenfeld and Axelrod (i), still seem to us, therefore, to be possib~7 explained by estrogenic con%~m~matlon of their norethynodrel dose.

REFERENCES i. Eisenfeld, A.J. and Axe lrod, J. J. Pharmaeol. and Exp. Therapeut. 150, 469 (1965). .

.

Jensen, E.V., Jaoobson, H.E., Flesher, J.W., Saha, N.N., Gupta, G.N., Smith, S., Coluooi, V., Shiplaooffe, D., Newmann, H.G., De Sombre, E.R. and Jungblut, P.W. Steroid D~,,=4cs. G. Pinous, T. Nakao and J.F. Tait, eds. A@a¢lemio Press, New York, pp 133-156 (1966). Stelnetz, B.G., Meli, A., Giannlna, T., Beaoh, V.L. and Nan-4~Z, J.P. Proo. Soc. Exp. Biol. and Msd. 124, ill (1967).

4. Golab, T. and Layne, D.S. J. Chromatog. ~ 321 (1962).

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