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A REVlEW OF SOME A P P L t E O HORHONE ASSAY METHODS R, J. OEANS

MI C H I GAN STATE U N I V E R S 1 TY ........ o.o.ooo...o...ooooooo.oo....oo..oo...o.-oo.o.~o.

Toda;y much is being writ ten and said about the use of hormonal substances i n c m e r c i a l lamb and c a t t l e feeding.

Their use has been cleared in implanting lamb with cambination progesterone-estradiol pe l le t s and incorporating diethyls t i lbestrol i n cattle feeds.

With t h i s widespread ccermercial we of these physiologically act ive substances it is necessary t o determine residual hormonal substances remaining in meat from animals having been administered these hormonal substances.

An assay is a technique either biological o r chemicd which is used to : (1) determine i f an unknown substance has hormonal character- i s t i c s , and aP w h a t type hormonal substance it is; (2) the re la t ive potency of t h i s hormonal substance, i.e., 8, quantitative assay.

Biological assays are based on known effects which these hormones have on certain body f'unctiona or tissue6. It is based on the premise tha t cer ta in concentrations of unknown substances given t o suff ic ient numbers of similar laboratory animals w i l l e l i c i t responses i n relationship t o potency. This response is then compared t o that of a known s t a n b r d t o determine re la t ive potency.

Chemical methods are based on the a b i l i t y of t h e chemist t o identify a corqpound and then t o determine quantitatively the amount of hormone present in an unknown.

Because of the sizeable task of reviewing a l l types of hormone assays, t h i s paper will deal with those most l i ke ly t o be encountered by one interested in livestock and meats.

There are a number of problems t o be encountered i n assaying a hormonal substance by a biological method.

1. The variation of laboratory animal not only between assays, but within assay groups constitutes a major problex. Most bioassay techniques have set for th type of animals t o be used, size, age, degree of maturit.y and quite often, s t ra in . Certain inbred s t ra ins of laboratory animals have been developed for assay purposes.

2 . Environmental conditions must be kept constant. This i s par- t icu la r ly important when assaying substances such as thyroxin where the assay is based on C02 output and O2 intake. must be caref'ully studied so as not t o introduce endocrine

Feeds

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stimulating substances i n t h i s way. been shown t o have effect on thyroid ac t iv i ty . Since endocrine interrelationships are so markedly important t h i s could cause variation in expected results.

Temperature and l i g h t has

Wen with highly inbred s t ra ins of animals held under ideal con- dit ions, we m y get abnormal endocrine functions i n cer ta in individuals.

The endocrine balance in l iv ing organisms i s a very delicate re- lationship and when disturbed, a number of changes usually occur i n other endocrine glands i n the same organism. of t h i s is the thyroid anterior p i tu i ta ry balance.

A notable example

Unknowns nzsy contain biologically act ive substances which are not ident ical with the t rue hormone substance. A crude extract w i l l contain 8 mixture of estrogen and other substances and the potency it exhibits w i l l depend on t h e method used and t h e nature of the extract .

According t o E$;mens, different estimates can be obtained by alter- ing such variables as: solvent used, spacing of injections, number of injections, e t c , "The influence of such variables is so great tha t (except under special conditions) It is impossible t o a t tach any precise meaning t o the resul ts of an assay unless It is known i n advance that the substance under tes t is i n pure form and is ident ical with the standard preparation i n chemical constitution.

At the present, t h e most sensit ive and accurate assws available Even with these, only a few are considered t o a re of the biological type,

be reliably accurate from a quantitative standpoint. tendency f o r the most sensit ive t e s t s t o be the l e a s t accurate. sensi t ivi ty and accuracy is yet t o be reached.

There is a strong Combined

In order t o understand b&er t h e characterist ics of the various types of assays and t o res l ize thelr limitations, specific assay methods should be examined.

Estrogen - Characteristics

Estrogens a re steroids having the basic steroid structure shown below.

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EstroGens induce estrus and stimulate secondary sex organs and sex They stimulate growth characteristics in normal and ovariectonized females.

of the uterine muscle and endometrium, motility of the uterus and fallopian tubes, increase capillary permeability in the uterus and vaeina, thickening of the vaginal epithelium and lowering of the pH of the vaglnal secretion.

They are necessary for mammary gland development and prepare rammary tissues for prolactin stimulation.

There are two general types, natural and synthetic.

The most potent natural type estrogen is the main ovarin estrogen. urine an8 is much less potent:

Thep estradiol form is

Another natural estrogen, estrone is found in urine, ox adrenal glands, and placentae. in vivo from estradiol. of .1 ug. of international standard preparation of estrone.

It is 1/4 - '-active as estradiol and is formed An international unit of estrogen activity is that

Estriol, a third natural estmgen is found in urine and is less &ive than estrone. VaSrues of 1:l to 250:l have been obtained for estrone/estriol ratio.

"The potency varies enormously with the test method."

Like estrone, estriol is formed in vivo from estradiol.

Estriol is excreted in the urine as sulfates and glucuronides.

The most prominent synthetic estrogen at the present is diwlstilbestrol

Diethyl stilbestrol

This subst>mce is highly potent orally as well as by injection.

Other types of synthetic estrogens are hexestrol, The potency of diethylstilbestrol when injected, l i e s between that of estradiol and estrone. and dienestrol, both of which have similar properties to diethylstilbestrol.

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faany synthetic estrogens and their estrogenic androgens are not themselves estrogenic but exert their effect after metabolic transformation in the body.

- Administration - and Utilization - of - Estrogens

The mode of administration of estrogenic substances has lately been of considerable significance .

The most useful vehicle for injection of natural or synthetic estrogens is an - oil. estrogens are rapidly absorbed and little prolongation of action is attainable by increasing the amount injected.

“When injected, even large excessive doses of most

Estrogens are readily absorbed in the intestine but when ingested, they exert relatively little estrogenic activity, owing to oxidation and conjue;ation in the liver. The liver can reversibly oxidize estradiol to estrone and to other products and it secretes estrone into the bile.

The rate of uptake of an injected hormone substance is a vital characteristic in its assay. to be injected in such are even more rapidly lost from the site of in- jection and excreted or destroyed.

Estrogens soluble enough in water or saline

The characteristics of the hormonal substance itself offset the rate of utilization. chain the more greatly prolonged was the effect.

According to Meischer (1) the longer the aliphatic

The minimum dose required to produce an estrogenic effect rises with the length of the aliphatic chain or chains, as shown by Parkes (2 ) . The mechanism by which esterification exerts its characteristic effects is by delaying absorp5ion fromthe site of injection.

When an injection technique is used, the potencies of the natural

This occurs with hydrolyzed extracts when multiple injections of estrogens most closely approach one another if absorption is slow, but not too slow. an oil solution are given or by the addition of palmitic or other fatty acids.

Substances siTj.1.ar to these fatty acids are present in some ex- tracts and may so equ.q,l.ize the potencies of the natural estrogens that use- ful and reasonably consis5ent results may be obtained by a two injection technique.

Estrogen Assq - Procedures As previously stated, estrogen causes cornification in the vaginal

cells. This principle is used in the Allen-Daisey Vaginal Smear Assay

Dosage: Solutions made up so that .OS to . 2 m l . is in- jected each time.

No. of injections: For routine assap - 2 injections are employed.

Smear: Smears are then taken 3rd and 4th dw. Three smears are taken each time. Test is considered

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posit ive if any of 3 smears is posit ive. All smears are taken from dorsal vaginal wall.

Staining : Smears are then stained with methylene blue.

I n t e r - e t a t i o n and Discussion of Data

cent posit ive slides

--- .-- -- -- - - Dosage expressed as logarithim should be plotted against per

Sample data tabulation and graph is shown below.

Per cent - Positive Respons 12 hrs. 24 hrs. 36 hrs. -- -I__

1

2

3

4

2.5

0

-2.5

10 .5 I.U. 0 0 71

10 1.0 I.U. 14 42 89

10 2.0 I.U. 14 89 100

0 0 0 l o - - - -

100

24 hour response plotted

Log Total Dosage (expressed i n gama or I . U . )

Probably one of the most accurate and sensit ive techniques f o r estrogenic assay is that set for th by Rubin-Dorfman (3).

The assay I s based on the increase i n weight of the immature mouse uterus and is conducted as follows:

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Mice are obtained 23 t o 25 days of age.

Animals are indected subcutaneously once daily for 3 da,ys with material suspended i n corn o i l . Daily dose was con- tained .1 CC. o i l .

Twenty-four hours a f t e r injection, uterine weights and bow weights were determined.

The uterine weight/body weight r a t i o was determined.

Four equal sized groups of animals were injected, two with a standard, two with an unknown.

The concentration of stambrd and unknown was chosen so that the followindy relationship held:

High dose s td . 1~ H i g h dose unknown LQW dose s td . Low dose unknown

With t h i s experimental design it is possible t o calculate:

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3. Mean slope ( )

4. Significance of difference between respective slopes.

5. Index of precision the smaller the value of , the

Relative potency of EUI unknown preparation.

Standard error of potency r a t io .

more the precision.

Most calculations were based on uterine r a t i o vs. log dose. However, with estrone, calculations are based on the log of uterine r a t i o vs. log of dosage.

This is necessary when dosages range is wide (.04 t o .32 m i l l i - grams). maximum response levels, because chances of sensi t ivi ty threshold variation may cause skewed results.

In general, it is better not t o assay too near the minimum or

The mouse uterine weight method has the advantage over vaginal smear methods of estrogen assay i n t h a t the resul ts axe obdective.

Sens i t i v i t x

The sensi t ivi ty of the mouse uterus is such tha t .4 micrograms of es t r in , .02 micrograms of estradiol, 2 micrograms e s t r i o l c8n be detected.

IntravaRinal Pellet Estrogen Assay

An assay technique by Albriewc. (3) has been established using a pelleted type hormonal substance.

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Blood pe l l e t s were made using 10 per cent sesame o i l as a "binder". e l ec t r i c fan at roan temperature. After drying, it is mixed with sesame oil and harmnered in to pellets weighing 40-80 mg.

Blood is placed i n cellophane tubes and suspended i n front of an

Separate pellets =de from serum or c e l l s can be prepared by centrifuging blood.

Pel le ts are then inserted into the vagina with a trocar and are completely dissolved i n 24 hours. This technique, while not widely used, offers poss ib i l i t i es f o r cer ta in phases of assqyrtng where the nature of the unknown might make it d i f f i c u l t t o administer i n other ways.

ProaesteroE

Characteristics of t he Hormone - -- This hormone is secreted by the corpus luteum and has been iso-

lated only f r m t h i s gland. pound, with full progestational ac t iv i ty . form. te ro l .

It is the only known, naturally occurring ccm- It can be produced i n crystal l ine

It has been produced synthetic- from the vegetable s t e ro l stigmas- It is excreted as pregnandiol.

Progesterone) Pregnandiol Sodium Glucuronidate . i Pregnandiol has no known hormonal action. cretion of pregnandiol follow closely changes i n the production of progesterone. However, pregmndiol and the excretory form of other related compounds such as desoxycorticosterone. t e r ia l i n the urine cannot be taken as en accurate measure of the amount of progesterone secreted

However, it i s an anaesthetic. Cbsnges i n ex-

Thua, the detection of t h i s ma-

There are synthetic substsnces which are of minor prac t ica l significance .

-- E t h a l testosterone is the most potent synthetic substance. (1/10 the ac t iv i ty of progesterone). jection, but i s weak both estrogenically and androgenicaly. androstenediol has about one-half the ac t iv i ty of ethiny:? testosterone.

It is as active by mouth as by in- Ethinyl

Since progesterone is not effective t o any extent when given orally, it cannot be incorporated in to feeds fo r laboratory animals. Thus, the assay of animal t i s sues for residual progesterone is quite d i f f i cu l t . In addition t o the problem of administration, we do not have such rela- t ive ly simple response mechanisms as uterine weights and vaginal smears i n

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assaying progesterone. development of the estrogen primed uterus and the maintenance of pregnancy. Within a few days after fertilization, or on artificial administration of progesterone, there is a rapid increase in cell division of the surface epithelial cells. This activity is accompanied by a downgrowth of glands and an increase in strcinal cell proliferation.

Physiologically, progesterone is responsible for the

A very sensitive sssrqy method for progesterone has been developed by Hooker (4) This technique is based on the principle that the endometrium of the mouse exhibits hypertrophy of the stromal nuclei when stinullated by progesterone. This technique I s currently being used on samples of meat from implanted steers on test at Michigan State.

Procedure :

The hormone is dissolved in sesame o i l . The standard mount ad- ministered was .0006 ml. Undesirable distension was produced if more W&S injected. In order to administer such small amounts, a micrometer syringe technique must be established.

Test animals used were adult female mice which had been ovarecto- mized 16 days before test. quite detailed. ventral section opened to expose the uterine horn.

The test substances are carefully injected into the uterus with the section being tied off to prevent leakage of the mterial. The animals are then killed 48 hours after injection. segment is removed snd fixed In Lavdowsky's fluid. sections 6 microns in thickness are stained with Harris' hematoxylin and Eosin.

A positive test I s described as follows: enlarged and appear smooth and ovaJ. in outline as conpared to shrunken nuclei and clumped chromatin granules, in non-treated cells. This appearance in =one of the s t r d cells is con- sidered positive.

The technique of administration is The animtrls are anesthesized, and then the mid-

The uterine Paraffin

Stromal nuclei are

The authors feel that the response obtained is specific for progesterone since 6 microerams of desoxycorticosterone, 48 micrograms of testosterone, .00075 microgram of estradiol or .6 ug. of estrone did not produce similar results, although there was some enlargement of nuclei.

Sensitivity:

The minimal effective dose (least amount which induces a positive response in any stromal nuclei) is said to be consistent3y ,0002 micrograms of progesterone.

This method could be adapted to a quantal assay in which 100 per cent positive responses could be expected with minimal effective dose.

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Progesterone - Chemical Assay: Edgar (5) has developed a method by which micro-quantities of

progesterone can be estimated in blood from the ovarian vein of sheep and from follicular fluid of Grmf'ian follicles and ovarian cysts. involves extraction and partition between organic solvents and the semi- quantitative estimation by chromatographic separation on filter pager. Amounts varying from 1.5 to .3 micrograms per m l . of fluid could be detected.

this technique, butmol extracts the sodium pregnanadiol glucuronidate from the urine. After careful separation and filtration the Na P.G. residue is dissolved in hot alcohol and dessicated. The amount present is determined by weighing.

The method

The best method according to Ehraens is that of Venning (6). By

Testosterone :

This is a male sex hormone classified as an androgen. Testosterone is the most potent androgen, being six times more potent than androsteroae. Androgens affect the following biological activities:

1. Metabolism

2. muscle rnass

3. electrolyte metabolism

4. enzyme concentrations of various muscles and glands.

Androgens of course, cause the development of secondary sex characteristics in males. of androgens and female sex hormones.

There is evidence of some similarity in effects

Transandrostenediol, dehydroisoandrostenediol, androstenedione, and testosterone produce uterus growth in the immature rabbit. androstenediol produces stratification and cornification of the vaginal epithelium in the castrate rat.

Trans-

Most of the androgen is derived from the testes. However, it is possible that these are androgenic substances secreted by the adrenals.

Admini st rat ion:

Androgens are ineffective when administered orally. Thus, making an assay of their potency or presence in carcasses more difficat. crystalline androgen is implanted subcutaneously, slow absorption and pro- longed effects are obtained.

When

Assay Methods:

There are a number of various assay techniques for androgens. The most successful are those using:

1. Comb growth response in fowl.

2. Seminar vesicle and prostate response in the rodent.

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Capon Comb Growth IC

Gallagher and Koch (7) developed a method using as a response too l the comb growth of capons. Five daily injections of testosterone unknown i n 1 ml. o i l are used i n a group of capons, while inJections of testosterone standard in lml, o i l are made i n another group of capons. From t h i s a characterist ic curve was developed, which used a dose-response relationship.

Bliss (8) elaborated on t h i s technique by running l i v e concentra- t ions of unknown and two concentrations of standard and then determining potency rat io . The t o t a l concentration of testosterone propionate used for each animal should be in the range of 20 t o 160 micrograms. A t least 32 animals should be used on standard and unknown. obtained when log dose plotted against l og response.

A l inear response i s

Enslens (9) applied testosterone direct ly t o combs. Obtained s t ra ight l i n e for log dose response.

Chick Comb Method -- Dorknan (10) injected chicks with hormone suspended i n o i l . ;&e

obtained a s t ra ight l i n e log dose - response relationship.

Seminal - Vesicle - Prostate Response Method

Principle: - Testosterone w i l l repair the damages t o seminal vesicles and prostate caused by castration.

Procedure:

Testosterone standard and unknown are dissolved i n corn o i l of which .1 t o .2 ml. are injected daily (each animEll receiving e q w amounts of o i l . )

Tota l dosage with testosterone should f a l l i n range of 0-5 mg. Androsterone has been administered up t o 30 mg. Variation i n length of treatment ranges from 8-10 days. body weights taken and seminal. vesicles and prostate removed.

The seminal vesicles are then weighed (with prostate o f f ) .

Rats a re kil led,

The log of the dose is then plotted against the gland weights.

Thyroxin

This hormone secreted by the thyroid gland is of v i t a l importance i n regulating t h e rate of energy exchange i n t h e body. by i t s effect on basal metabolic rate and i n oxygen uptake of t i s sue s l i ce s .

It can be observed

Among the physiological characterist ics with which thyroxin i s associated are:

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amount of adipose tissue

rate of glucogeneisis

rate of excretion of urinary nitrogen

rate of in tes t ina l absorption of oxygen and i n t e s t ina l peristalsis.

respiratory quotient

levels of blood l i p ids

s tore of body proteins

storage of l i v e r glycogen

leve l of blood sugar

rate of secretion of urinary H20

rate of excretion of urinary calcium and phosphorus.

On the endocrine system, thyroxin affects :

1. secretion of thyrotropic hormone from the anter ior p i tu i ta ry

2 . secretion by adrenal cortex by ACTH stlmulation

3. conditioning of reactive parts of body epinepherine

4. appears necessary fo r adequate secretion of p r o l a d i n and consequent milk secretion.

There are a number of asssy methods available which apply some of the above Characteristics in response characterist ics.

Goiter Prevention Method

Principle - when a goitrogenic substance (thiouracil) which inhibits thyroxin production is given, we get increased thyrotropic hormone secretion and resultant hypertrophy of the thyroid,

This thyroid hypertrophy can be prevented by administering thyroxin t o restore n o d belance.

Procedure :

100-200 gram male rats are grouped (8-10). sufficient t o establish a response curve . l p e r cent thiouraci l i n feed during treatment period. (Dosage should be given t o f a l l In middle of response curve i f possible.) AU. but one group of thiouraci l t reated rats are then injected with 1-4 microgrems/100 g. body weight of thyroxin.

Five groups 4re usually All groups are given

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l6

14

l2

10

8

m$100 g. body w t .

After 14 days, thyroids are removed and weighed. Response is An i l l u s t r a t ion of reported as micrograms thyroxin/100 grams body weight.

graphic presentation i s as follows:

\ f \

Normal thyroid weight \\ -----‘t-- - -V

Note: when curve intercepts normal weight l i n e - can be taken as measure of thyroid secretion rate since it represents the amount of DL- thyroxin required t o maintain normal thyroid p i tu i ta ry balance.

Metabolic L- Rate Method

by i ts effect on the metabolic rate of guinea pigs. Dressler and Halling (11) reported a method of assaying thyroxin

Procedure : _I_-

Feed and water are withheld from guinea pigs fo r 18 hours before determination. Oxygen consumption and CO2 output is then deter- mined with metabolism being expressed as ml. 02 or C02 exchanged per 100 g. body weight per hour. These control readings are taken on 3 separate days.

Thyroid preparation is administered oral lx for 4 consecutive days. One hundred hours after the first dosage B.M.R. deter- mination is made and percent increase noted for each animal over control value.

A reference curve i s estimated by graded dosage with standard thyroid powder. computed in terms of the standard.

The thyroid equivalent can be established by reference t o standard slopes

The percent increase in oxygen consumption is substituted for y i n the equation. logarithm of thyroxin dosage that must be given oral ly t o give the same response.

The potency of the test preparation i s then

Then the equation is solved for X - t o get

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42

36

30

24

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$ increase

i n

oxygen consumption

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Thyroxin dosage ug/day/lOO g. body w t .

In the use of this type of assay, the reference curve should be established for a standard substance 'similar to those being tested.

BIBLIOGRAPBY

1. Meischer, K. Wettstein, A. Tschoppe, 1936. Biochemical J. 30, 1977.

2. Parkes, A. S. 1937. Biochemical J. 31, 579.

3. Uerieux, A. S. J. Clin. Endocrinology 1. pp. 889.

4. Hooker, C. W. 1940. Proc. SOC. Exptl. Biol. Xed. 45, 270.

5 . Edg8r, P. G. Prog. in Bcdy Fluids. Nature 170: 543. 1952.

6. Venning, E. H. Gravimetric method for the determination of sodium pre- gnandiol glucuronidate. J. Biol. Chem. ll9:473. 1937.

7. Gallagher, T. F., Koch. F. E. J. Pharm. Exptl. Therapy. 55: 97. 1935.

8 . Bliss, B. I. 1944, J. h r . Statis, Assoc. 39: 479.

9. Emmens, C. W. 1929. Med. Research Council. (Brit.) Special Rept. Ses, 234,l

10. Dorflnsn, I. Endocrinology 42, 7 1948.

11. DmeBLer, E., and U h g , K. 1940. Arch. Exptl. hth. Phamcology 196, 266.

12 EhPPens, "Hormone Assay"

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MR. A U " : We sure enJoyed the topic tha t you presented, and certainly when you cam t o some of the chemical compounds it was more enjoyable.

A t t h i s time we w i l l ask H. D. Naumann t o lead the discussion.

IVAUMA": Thank you, M r . Chairman.

The recent popularity of the use of hormones has caused quite a good deal of concern in the glace I am working. The physi- ologists and the endocrinologists are pret ty much up i n the a i r about it becauae from the standpoint of public health it could be a r e d problem if soll~ething got out of control. They are part icular ly ques- t ioning i n t h e i r own minds the val idi ty of many of the assay technics that are being used today framthe standpoint t ha t a l o t of them are biologicst assqys and as a result they are used on tes t animals and there isn't def in i te proof that the response of the tes t animal is analogous t o the response of the human with whom we are ultimately concerned. The other question i s perhaps j u s t as important i n that i n many of these products the hormones specifically seem t o have a cumulative effect or at least that is a strong poss ib i l i ty . So the reviews of hormone assw methods i n qy opinion are pertinent at the moment.

Do you have any questions t o direct t o the speaker?

W E L I C : I should l ike t o make a comment about the point of the amount of a substance, whatever it may be, that may be present i n t i s sue . a l l have recently, concerning how l i t t l e there may be of a given sub- stance in meat. quantitative amount is not the most important thing. The most im- portant thing is what amount will produce physiological effects.

I have seen reference i n several reports, as you

I think we should &e it absolutely clear that the

I should l i k e t o drive home t h i s point by pointing out that it mtters l i t t l e if it takes a fract ion of a gram of a biologic substance t o kill a person and it takes several pounds of sodium chloride. That is one point I should l ike t o put across.

If t h e end result is fatal, it matters not how much it took.

I think, too, there has been a dangerous point i n the con- clusions drawn i n some of these experiments. that one observes when he deals with a short-term experiment o r an acute toxici ty test. effects than I am about acute, because i f we can produce effects immedi- ately that are syqptomaticslly obvious, we are warned, but there are instances *ere it may take years, indeed, before the results of the ingestion or the use of acme rtmterialmight come about and be recog- nizable as such. express the most concern; y e t I hear people with pract ical ly no temerity whatsoever saying, as the resu l t of their assays, tha t there are not l ike ly t o be any effects . the remarks Mr. Dew made he brought forward same of these things

That concerns the effect

Personally I am far more concerned about chronic

It is i n t h i s lat ter category t h a t I think we should

I think tha t i s naive, and I am glad that i n

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Another th ing tha t I th ink we should be concerned about ( th i s is my t h i r d point) is that i f by one cr i ter ion you produce no evidence of effect tha t does not preclude the possibi l i ty that there are no effects . weight of the mouse i s frequently used t o establish whether or not the material my be present. That I s one thing, but the animal organism is extraordinarily complex and there may be other effects.

I n the case of the s t i l bes t ro l assay the uterine

I am not an alarmist. I want t o make that clear, but le t ' s be a l i t t l e thoughtful about some of these assays before we write some of the things tha t have been written.

EIR, BULL: M r . Chairman, is it possible t o hook a hot carbon on these estrogens that are fed the animal? done?

If so, has it ever been

MR. BVTLER: The question?

MR. Can you use a t racer technic on these hormones? Who would care t o answer t h a t question?

MR. KASTELIC: of carbon i n diethyls t i lbestrol . been the ethyl groups. has been indicated from such studies tha t the material is not metabo- l ized I would not be satisfied with such information u n t i l the r e - maining part of the molecule had been tagged and u n t i l the degradation products, if any, are carefully mapped out. molecule i s not suff ic ient .

There are some papers concerned with the tagging The carbon tag i n these instances has

We a l s o have two-ring structures, and while it

Tagging one piece of a

MR. BULL: In connection with D r . Kastelic's remarks about long-time effects, there is rather prevailing sentiment among medical men that the use of estrogen by the ladies i n face cream, bust devel-- opers, etc., causes cancer. The same is t rue about the use of tes tos- terone among you old fellows.

MR. "N: Do we have other comments or questions?

Well, I want t o take the l i be r ty , since Deans has mentioned th i s i n h i s paper, of directing a question t o him, although perhaps it i sn ' t real iy within the scope of the t i t l e . .What were the results of these progesterones on meat t issues?

MR. DEANS: On our last bunch we did not get any a t a l l . We The thing we are com- have different hormone treatments a t t h i s t i m e .

paring here is an implant as w e l l . have different hornone dosage. and predict w h a t we are going t o get.

O f course, we have estradiol . So we don't want t o go out on a limb

We

We didn't get any the last time.

Any other questions? Then I will turn it back t o the Chairman.

MR. AUWW: of the University of I l l ino is .

A t t h i s time we w i l l hear from D r . Breidenstein

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