K. E. Paschkis: I have one question and one comment. The question is with regard to localization. You didn't have the breast tissue on your slide; did you investigate how much of the injected dose goes to the breast? The comment is apropos Dr. Engel's remark, questioning whether small amounts, physiological amounts, also appear in the bile in large part. Many years ago (before the era of radioactive labeled compounds), we studied biliary excretion of estrogens and found we had to give large doses of estrogen in order to get any biological activity in the bile which we could measure. We were criticized as not dealing with a physiological mechanism when administering these huge amounts of estrogen. Consequently we gave gonadotropins and forced the ovary itself to put out estrogens; again there was a very high biliary excretion. I wouldn't be at all astonished if in Dr. Jensen's animals the estrogen were in the bile.

E. V. Jensen: In answer to your question about breast, this is a very interesting story which we hope to be able to say something about in time, but I am not prepared to do so at present. In regard to your comment, we would be very happy to be able to account for the rest of the radioactivity in the bile. From our standpoint, though, accounting for the total radioactivity is of secondary interest, because our primary objective is to determine what is happening to the steroid in the "target" tissue and, further, how this differs from its fate in "nontarget" tissues.

M. M. Mason: I was interested in your report of anti-uterotrophic activity. You showed the results with only one compound, and the order of inhibition was about 60%. We have tested to date about 300 or 400 compounds and we have about 100 compounds that show anti-uterotropic effect over 50%. We would like to know whether any of the nonsteroidal compounds show the same mechanism. It would be interesting in the investigation of anti-uterotropic activity to find out where this blocking takes place. With this very elegant system you have evolved, residual hormone might very well show where this blocking takes place, and whether the estrogen is still present in the target tissue despite the lack of growth.

E. V. Jensen: We have looked only at fluoro hydroxy progesterone in this experiment, since, in an investigation of estrogen inhibition that Dr. Huggins and I carried out and reported in 1955, this was found to be the most effective inhibitor studied. Obviously this is just a preliminary approach to the mechanism of hormone antagonism. One should measure not only how much radioactive estrogen reaches the uterus with and without the administration of inhibitor, but also whether differences exist in the nature of the radioactive material present in the tissue. But since it now appears that in the absence of inhibitor the active material in the uterus is unchanged estradiol, it would be rather unexpected if the inhibitor caused the uterus to convert the estradiol into something else. But we shall have to do more along this line, as you suggest.

G. C. Mueller: Dr. Jensen, you certainly have filled in a tremendous gap in the information that we have wanted for a long time; that is, the state of hormones in the tissue during response to hormone. This beautiful work is an example of experimentation executed with good command of organic chemistry and good knowledge of the biological picture. I was wondering, however, if you would care to disclose something of your techniques in radioactive counting. In our experience it can be very difficult with some of these tritiated substances.

E. V. Jensen: This presented quite a problem when we began, since there was no simple satisfactory method for the determination of tritium in biological material. The difficulty lies in the very low energy of the beta radiation from tritium which renders end-window or even gas-flow counters inadequate.

We have developed a procedure in which the tritium of the sample is converted to water, which is then counted in an automatic liquid scintillation counter. In this method, the dried tissue sample is heated at 650° C. with copper and copper oxide in a sealed tube of special glass, according to a procedure described by Wilzbach and Sykes some years ago for the determination of isotopic carbon in organic compounds. This treatment converts the carbon to carbon dioxide, the nitrogen to molecular nitrogen, and the tritium and hydrogen to water, which is easily condensed from the other gases in a vacuum manifold and transferred to a counting bottle. Although several manipulations are required for each sample, the fact that you can process a large number of samples simultaneously renders the method suitable for routine assay. Using this combustion technique, there is no interference in the scintillation counting from colored or quenching substances present in blood and tissues. So far as I know, this is the only method that we possibly could have used for the large number of determinations that we have carried out. Description of this method should appear in the Archives of Biochemistry and Biophysics before the end of the year.

Y. J. Topper: I should like to comment further on the question of steroid responsiveness of so-called "target" organs and "nontarget" organs. Some time ago we reported on observations indicating that progesterone had a considerable stimulatory effect on the oxidation of galactose by liver in vitro. The only other tissue in the mammalian system which we found responsive in this respect is gut. In order to see whether there was any physiological counterpart to this in vitro effect we made a study of three galac-tosemic children. Galactosemia is a disease characterized by an inability to metabolize galactose completely. This is a consequence, as was beautifully demonstrated by Kalckar and his associates, of an enzyme deficiency. The enzyme, galactose-1-phosphate uridyl transferase, is missing in these individuals. We were able to show that, whereas these children are essentially incapable of metabolizing galactose to carbon dioxide during a control period, after several days of progesterone administration their ability to metabolize a tracer dose of galactose-approaches that of the normal subject. I might add that following progesterone treatment the blood cells derived from these individuals are still incapable of metabolizing galactose. It appears that the liver, an organ not usually considered to be a target organ for progesterone, has been able at least partially to circumvent the enzymatic block in response to this hormone.

G. Pincus: I would like to continue with this idea of a target organ. I think that there is scarcely any tissue in the body which is not responsive to estrogens. I had the opportunity of reviewing this some time ago, and as far as I recall the eyeballs are not affected by estrogens. Every other tissue has been recorded in one way or another to respond to estrogens, sometimes very surprisingly, other times rather superficially. So I am wondering whether it is quite correct to speak of the uterus and vagina as "the" target organs.

E. V. Jensen: The original reason for picking on uterus and vagina is because these are tissues which grow spectacularly in response to the estrogen stimulation. Growth response could be one basis of assigning the term "target organ." I have tried to point out that such an assignment could be based on two other criteria, namely, the shape of the steroid incorporation curve and the chemical nature of the radioactive material that is present in the tissue. In fact, according to the latter criterion, one might have to define a target organ as one which doesn't do anything to the steroid, since in uterus the radioactive material seems to be free estradiol, whereas in a tissue such as liver something does happen to the steroid. I certainly agree with Dr. Pincus that estrogens must affect most, if not all, the cells of the body. Yet, no matter what one calls the different types of organs, I think one should distinguish tissues such as uterus and vagina from others such as liver, kidney, and muscle. This is what I tried to bring out.

S. Kushinsky: I should like to ask Dr. Jensen (1) if he has examined fatty tissue for radioactivity content, (2) the location of the sample of muscle which was analyzed, and (3) the site of injection of the radioactive material.

E. V. Jensen: I'll answer your last question first. The material was injected sub-cutaneously in the back. The muscle was a thigh muscle, the M. Quadriceps femoris. In regard to the fatty tissue, we have no data on this as yet.

V. P. Hollander: Since the estrogen-stimulated rat uterus is hyperemic and has a high water content, I would like to know how much radioactivity is associated with the tissue fluid and how much with solid residue.

E. V. Jensen: The uteri in cur experiments are wet but not bloody. These are rather atrophic uteri from castrate or immature rats. The uteri taken 2-6 hours after estrogen administration have grown a little; they are slightly bigger than the controls on both the dry and wet weight basis, but they are not bloody. There is a little bit of luminal fluid present, and of course, in the 7-day experiments, there is a lot of luminal fluid. In one 7-day experiment we measured the radioactivity in the luminal fluid and, somewhat to our surprise, found it to be very low. In the tissue water, obtained on drying the uteri for assay, there is definitely a little radioactivity, but this volatile material is very small compared to the nonvolatile.

U. Kim: It is interesting to note that Dr. Jensen's work included the anterior pituitary gland as one of the target organs of estrogens. It is our belief that estrogens stimulate mammotropes of the anterior pituitary to produce hormone or hormones which, in turn, stimulate the mammary gland. In other words, estrogens are mediated by pituitary to act on mammary tissue. We think this concept can be substantiated by the classical experiments of pituitary tumor induction by chronic administration of estrogens in rats. Dr. Furth and his associates have demonstrated the function of such tumors. We also observed that, in absence of pituitary, estrogens failed to give mammary gland stimulation. This phenomenon was also observed in hormone-responsive mammary tumors. By grafting functional mammosomatotropic tumors we have been able to revive mammary tumors which had regressed following various endocrine ablative therapies.

E. V. Jensen: I agree with you that the question of the breast is very important, and it is one that we are studying. However, one is limited in how much he can do at a time, and as yet I don't have a story to tell about this.

C. A. Villee: I was struck by the fact that when you inject estradiol you recover essentially only estradiol. However, when you inject estrone you recover quite a mixture of things. This seems very curious to me. I wonder if you would like to comment on that?

E. V. Jensen: This is in the uterus. I would like to be able to say more about estrone because it is a very interesting question. Whereas most of the other experiments reported have been repeated and confirmed, this one with estrone represents a single experiment which needs repeating. The various radioactive fractions must be rechro-matographed and positively identified. What presumably is the estradiol fraction is somewhat larger than any other fraction, but there certainly appears to be a variety of substances present. At this time, we can say only that this gives a preliminary indication that estrone is different from estradiol, and it may be that the biologically active agent is estradiol into which part of the estrone is metabolically converted. We shall just have to look further into this phenomenon.