As discussed in some detail by Dr. Drill, it is well established that certain steroids can exert a marked inhibitory effect on the physiological actions of other steroids. One such example is the ability of progestational hormones to inhibit the uterine and vaginal responses to estrogens (1, 5, 9). In a study which Dr. Huggins and I carried out a few years ago (6), 9a-fluoro-11β-hydroxyprogesterone (FHP) was particularly effective in this regard. Conceivably, the FHP might antagonize the estrogen either by preventing its incorporation into the responsive tissue or else by interfering with its action at the receptor level. As a preliminary approach to this problem, we determined the amount of radioactivity incorporated into the uteri both of immature and of hypophysectomized rats following seven daily subcutaneous injections of 0.1 pg. of tritiated estradiol in sesame oil, with and without the simultaneous injection of 1.0 mg. of fluorohydroxyprogesterone. As seen in Table V, in the immature rat the inhibited uterus is about half as large as the noninhibited uterus (on either a wet or dry weight basis), and incorporates about half as much radioactive steroid. A roughly similar pattern is seen with the hypophysectomized animals. One might conclude that the inhibited uterus grows only half as big because the FHP reduces by a factor of two the amount of estradiol incorporated, or one may argue that, if the total amount of estradiol taken up depends on the size of the organ, the similar concentration of radioactivity in both cases signifies that the inhibitor has no effect on estradiol incorporation. But no matter how one wishes to interpret the result, it is interesting that the rat uterus incorporates half as much estrogen and grows to half the size when the inhibitor is present.

Table V. Effect Of 90-Fluoro-11β-Hydroxyprogesteiione On Incorporation Of Estradiol Into Rat Uterus*

* 0.1 µg. Estradiol (20µc./ug.) and 1.0 mg. FHP administered daily for 7 days by subcutaneous injection in sesame oil. Tissues assayed on eighth day. Each figure is the mean value of samples from six animals.

Summary

Tritiated estradiol and estrone have been prepared with specific activity sufficient to permit measurement of 1µµg. of steroid; a method has been developed for the routine assay of tritium in animal tissues. These tools have made possible the accurate determination of the amount and nature of radioactive steroid present in various tissues of the immature rat at different time intervals after the administration of physiological amounts of steroid estrogens. Two to six hours after the injection of 0.1 µg. of estradiol, the amount of steroid present in the uterus is of the order of SO to 150 µµg.

Uterus and vagina differ from other tissues, such as liver, kidney, muscle, adrenal and blood, in that the radioactive steroid reaches a higher concentration and continues to be incorporated and retained in these growth-responsive tissues at a time when the radioactivity in the other tissues has declined to a low level. In uterus the major portion of the radioactive material is free estradiol, with practically no water-soluble or protein-bound activity being observed, whereas in liver the free steroid appears to be a mixture of substances, and, in addition, appreciable protein-bound and water-soluble radioactivity is present. Thus, both by steroid incorporation pattern and by the chemical fate of the estrogen in the tissue, one can distinguish between two types of tissues, illustrated by uterus, on the one hand, and by liver on the other. Whether these should be designated as "target" and "metabolizing" tissues, respectively, is a matter of individual opinion, but the phenomena here described, in addition to the classical growth-response behavior, afford criteria for the clear differentiation of the two types of tissues.

References

1. Dessau, F. Acta Brev. Neerl. Physiol. Pharmacol. Microbiol. 7, 126 (1937).

2. Emmens, C. W. J. Endocrinol. 2, 444 (1941).

3. Fishman, J., Bradlow, H. L., and Gallagher, T. F. J. Am. Chem. Soc. 81, 2273 (1959).

4. Glascock, R. F., and Hoekstra, W. G. Biochem. J. 72, 673 (1959).

5. Hisaw, F. L., and Lendrum, F. C. Endocrinology 20, 228 (1936).

6. Huggins, C., and Jensen, E. V. J. Exptl. Med. 102, 347 (1955).

7. Hurlock, B., and Talalay, P. J. Biol. Chem. 227, 37 (1957).

8. Jacobson, H. I., Gupta, G. N., Fernandez, C., Hennix, S., and Jensen, E. V. Arch. Biochem. Biophys. 86, 89 (1960).

9. Korenchevsky, V., and Hall, K. J. Pathol. Bacteriol. 60, 295 (1940).

10. Muhlbock, O. Acta Brev. Neerl. Physiol. Pharmacol. Microbiol. 10, 42 (1940).

Discussion

C. A. Villee: I have one question to ask before the general discussion: Do those numbers in your table on the blackboard represent the percentage of the total injected dose recovered in each tissue?

E. V. Jensen: Yes.

C. A. Villee: So you have accounted for a little more than 2 % of the total dose. Would you comment on where the rest of it is?

E. V. Jensen: The only indication we have is that a considerable part of it is in the kidney and urine. In this experiment we do not have the kidneys assayed yet, but from previous experiments we know the kidney is relatively rich, and we know qualitatively that the urine is also.

I think that this rapid falling off of the blood and liver levels signifies a rapid clearance of the administered hormone, even though we administer no more than what we think is a physiological dose. The actual physiological dose appears to be much less than that administered, and a major part of the radioactivity may appear in the kidney and urine, and also in the enterohepatic system, which we have not yet studied.

A. A. Sandberg: This might clear up the point for Dr. Villee: within 10 minutes after the injection of small amounts of C14-estrone we have found nearly 90% of the radioactivity in the bile of the rat

E. V. Jensen: This might very well be the case in our rats, and it is something we will have to look into. Thank you for clearing up this point.

L. L. Engel: It may be hazardous to draw the conclusion that the behavior of a relatively large dose of the steroid is the same as that of the extremely minute dose given here. It may well be that the biliary excretion is an overload phenomenon when physiological limits are exceeded.

S. Solomon: In your hydrogenation with tritium gas, how much nonspecific exchange is there? This is important to know in view of the minute amount of tritium that is measured in the tissues. The point that Dr. Engel just made is substantiated by the experiments done in mice by Dr. R. D. H. Heard and his collaborators in 1951. They injected 1 mg. of estrone-16-C14 per mouse intramuscularly and found C14O2. When these experiments were repeated by others using much smaller doses of labeled estrone, no C14O2 was detected.

E. V. Jensen: In answer to your question about the preparation of the material, both we and Dr. Wilzbach, with whom we have discussed this, feel that there is very little exchange during this tritiation. As you know, Wilzbach exchange is carried out by exposing an organic compound without any catalyst to carrier-free tritium gas usually over a period of at least a week and one gets random or semirandom labeling with tritium. Our tritium gas is removed after 15 minutes, so the opportunity for Wilzbach exchange is quite limited. Secondly, the specific activities obtained by Wilzbach exchange are many hundred times smaller than what we have here. Therefore, even if exchange took place in these 15 minutes, the tritium thus introduced should be negligible in comparison to that resulting from double-bond reduction.