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Estrogenic Residues in the Edible Tissues of Stilbestrol-Fattened Chickens
118
P. B, SlEGEL
CONCLUSIONS
Significant differences among lines of White Plymouth Rock cocks were found for aggressiveness and sex drive, indicating a genetic basis for these traits. No heterotic effect, with regard to these characteristics, was exhibited when cross line cocks were compared with cocks from the
inbred lines from which they originated. Correlations between the behavior traits studied indicate a positive relationship between aggressiveness and mating ability. Correlations among the 4 types of mating behavior measured, indicate that males which exhibit the largest number of courts, mounts, and treads, also complete the largest number of matings. REFERENCES Guhl, A. M., 1953. Social behavior of the domestic fowl. Kansas Agr. Expt. Sta. Tech. Bui. 73. Guhl, A. M., 1956. Personal communication. Guhl, A. M., N. E. Collias and W. C. Allee, 1945. Mating behavior and the social hierarchy in small flocks of White Leghorns. Physiol. Zool. 18: 365-390. Lerner, I. M., 1954. Genetic Homeostasis. John Wiley & Sons, Inc. New York. Valenstein, E. S., W. Riss and W. C. Young, 1954. Sex drive in genetically heterogeneous and highly inbred strains of male guinea pigs. J. Comp. Physiol. Psychol. 47: 162-165. Wood-Gush, D. G. M., 1956. The agonistic and courtship behavior in the Brown Leghorn cock. Brit. J. An. Behav. 4: 133-142. Wood-Gush, D. G. M., 1957. Aggression and sexual activity in the Brown Leghorn cock. Brit. J. An. Behav. 5: 1-6. Wood-Gush, D. G. M., and R. Osborne, 1956. A study of differences in the sex drive of cocks. Brit. J. An. Behav. 4: 102-110.
Estrogenic Residues in the Edible Tissues of Stilbestrol-Fattened Chickens ERNEST J. UMBERGEE, GEORGE H. GASS, KENT J. DAVIS, JACK M. CURTIS AND CHARLES G. DURBIN
Food and Drug Administration, U. S. Department of Health, Education, and Welfare, Washington, D. C. (Received for publication May 23, 1958)
T
HE use of synthetic estrogens for the hormonal fattening of poultry, and studies designed to demonstrate whether or not estrogenic residues remain in the
edible tissues of the treated poultry have recently been reviewed by Lorenz (1954) and by Merker et al. (1955). Commercial use of the estrogens has been limited to the
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not always lower than that of the heterogeneous stocks. The significance of pen effects points out that experimental design is important in testing for sex drive. Pen effects were much larger than had been anticipated. If all males had not been tested with all females, much of the information obtained in this study would have been invalid. The barely significant correlations between encounters won with number of courts and completed matings support the thesis of Wood-Gush (1956) that there may be a relationship between aggressive elements and courtship displays. However, the non-significant correlation between aggressiveness and both mounts and threads indicates that this relationship is probably minor. The highly significant correlations between the four types of mating behavior measured show a high degree of relationship among them.
ESTROGENIC RESIDUES IN TISSUE
EXPERIMENTAL
Assay Methods. The details of the bioassay method and the experimental de-
signs are described in a paper by Umberger, Gass and Curtis (1958). In general, the method consisted of grinding 100 parts of tissue with 10 to 20 parts of laboratory diet and feeding these diets to immature female mice for seven days. On the eighth day the animals were sacrificed and their uteri weighed. Each animal received 6 or 7 grams of diet per day, an amount which was fully consumed and permitted some growth. Usually, six mice at each dosage level were used. To obtain a quantitative measurement of added estrogenic activity in the treated tissues, and at the same time to estimate the sensitivity of the method, three experimental designs were used. In the first design, stilbestrol was added to both control and treated tissue diets fed to the mice at levels of 0, 2, 4, and 6 parts per billion. At these very low dosage levels, a linear relationship exists when the dose is plotted versus the log of the ratio of the uterine weight in milligrams to the final body weight in grams. Any displacement of the treated tissue curve away from the control curve along the response axis measures the amount of estrogenic residues in equivalents of stilbestrol. When the level of estrogenic activity was greater than 2 parts per billion, a second design termed "the rangefinder" was used. This consisted of feeding control tissue diets, to which had been added 0, 3, and 6 parts per billion of stilbestrol, to three groups of mice and at the same time feeding treated tissue diets, diluted with 0, 50, 75, 87.5, and 93.75 percent control tissue diet, to five other groups of mice. By proper selection of responses, the data could be calculated as a balanced slope-ratio assay, or the data could be used to estimate graphically a dilution factor for use in the third experimental design. In this third design the treated tissue diet was diluted with control tissue diet so that the expected
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t
implantation or injection of stilbestrol and to the oral feeding of dienestrol diacetate. The usual practice is to treat the birds at 7 to 9 weeks of age and to slaughter the birds 4 to 6 week later. With the development of a sensitive assay method (Umberger, Gass and Curtis, 1958) based on the work of Stob, Andrews and Zarrow (1954), an examination has been made of the edible tissues from chickens treated with various commercial preparations of stilbestrol. The results of these assays are the subject of this report. A subsequent report will detail the studies with dienestrol diacetate. The literature contains references to both biological and chemical assay methods for the detection and estimation of estrogenic residues in tissues of poultry following stilbestrol treatment. Table 1 summarizes in chronological order all the references available to us using biological assay methods. It will be noted that data are available on both chickens and turkeys, and that with one exception the method of treatment was by pellet implantation. We note also that in only two of the ten cases were the tissues fed to the assay animals; the other assays involved solvent extraction of the tissues and subcutaneous injection of the extract dissolved in oil. Studies involving chemical assay for stilbestrol in tissues were those of Jones and Deatherage (1953) and Swift (1954). The former were unable to detect any estrogen in edible tissues, but did find residues at the site of injection of a 12-mg. pellet or a 15-mg. paste preparation. Swift (1954) found evidence of small amounts of stilbestrol in extracts of liver and skin, but not in the muscle of cockerels treated with a 15-mg. pellet of stilbestrol.
119
12 mg. pellet
Chicken
Turkey
Campbell et al. (1952)
Greenblatt et al. (1952)
Koch et al. (1952)
Stob et al. (1954)
Snair et al. (1954)
Merker et al. (1955)
5
6
7
8
9
10
12 mg, pellet
12 mg. pellet
12 mg. pellet
20 mg. pellet
15 mg. pellet 30 mg. pellet
12 mg. pellet
Carcass
Fat
Carcass
Muscle Bones Skin Liver and gall Abd. fat
Fat
Carcass Liver Liver
Carcass Liver
Liver
* DE—Stilbestrol equivalents. f EE—Estrone equivalents. X Authors also calculate in DE using factor 0.08/0.06.
Chicken
Chicken
Chicken
Chicken
Chicken
12 mg. pellet
Chicken
Meyer (1952)
4
15 mg. pellet 30 mg. pellet
Chicken
Colombel (1950)
3
Abd. Fat Liver Fat Skin-muscle
48 mg. pellet
Turkey
Gowe (1949)
2
Abdominal Eat
10 mg. in oil
Stilbestrol preparation
Chicken
Species
Bird et al. (1947)
Investigators
1
No.
Tissue assayed
Vaginal Smear Vaginal Smear
Mouse Castrate Mouse Castrate
Ext.-chloroform Ext.-chloroform
s.c. oil
Oral s.c. s.c. oil
Uterine Weight Vaginal Smear
Uterine Weight Vaginal Smear Uterine Weight
Mouse Immature Mouse Castrate
Mouse Castrate Rat Castrate Mouse Castrate
Ext.-ether
In food Rendered Ext.-ethanol, ether, alkali, ether
s.c. oil
s.c. oil
s.c. oil
Oral
s.c. oil
s.c. oil
Route
Ext.-benzene-ethanol
Vaginal Smear
Rat Castrate
In food
Vaginal Smear
Vaginal Smear
Response
Rat Castrate
Rat Castrate
Animal
Method of assay
Ext.-methanol, ether
Ext.-ether
Preparation of sample Results
Absent
Present
Present
Present in all. From 0.01 to 34 mg. per 2,200 g. bird
Absent
Absentf 60-118 ppb. E E 50 ppb. E E
13+ 3 p p b . E E f 24±19ppb. EE
210 jug. per 35 g. liver (6,000 ppb. DE*)
Absent Present Present
Absent
of the literature on the biological assay of the edible tissues of stilbestrol-treated poultry for estrogenic residues
Treatment
TABLE 1.--Summary
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121
ESTROGENIC RESIDUES IN TISSUE
Pellet: A 15-mg. pellet containing 12 mg. of stilbestrol. Paste: A preparation of paste-like consistency having a water-soluble base and a wax-like substance to delay absorption of the stilbestrol. Liquid-Rosin: A preparation of liquid
consistency having a water-soluble base and gum rosin to delay absorption. Liquid: A preparation of liquid consistency having only a water-soluble base. Experiment No. 1. 400 New Hampshire cockerels were divided into 16 groups of 25 each. Eight of these groups were kept for controls; the other 8 groups were treated with the four preparations containing two dosage levels of stilbestrol. The birds were treated at 8 weeks of age and were slaughtered 5 weeks after treatment. The following tissues and organs were saved: Eviscerated carcass, giblets (heart, gizzard, and liver), offal (small intestines, split and washed), and heads (from treated groups only). Abdominal adipose tissue was not saved since the only group which had any appreciable amount was the pelleted group, and therefore, sufficient control tissue for an assay was unavailable. Tissue assays were made on lean meat trimmed from the eviscerated carcasses, on the giblets, and on the offal. The results are shown in Table 2. In this experiment the tissues were fed raw. It will be noted that no estrogenic residues were found in T A B L E 2.—Experiment No. 1. Estrogenic residues the tissues of chickens 5 weeks after treatment with stilbestrol (DES) Treatment
in
Residual estrogen in DES equivalents (ppb.)
mg. DES
Vehicle
Lean*
Giblets
Offal
12 15 15 15 6 7.5 7.5 7.5
Pellet Paste Liquid-Rosin Liquid Pellet Paste Liquid-Rosin Liquid
0.8±0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0
>6 >6 >4 0.0 >6 2.0
>5 >5 0.8+0.4 0.0
-t
0.0
0.0=Level of estrogenic activity not significantly different from controls. t=Significant estrogenic activity indicated but not quantitatively determined. > =Level of estrogenic activity too great for calculation. Figure shown is a minimum value. * = Consisted of both leg and breast muscle, skin removed.
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•
concentration of estrogenic activity in stilbestrol equivalents was between 1 and 2 parts per billion. The control tissue diet and the diluted treated tissue diet were then spiked with 0, 2, 4, and 6 parts per billion of stilbestrol as in the first experimental design. When there was insufficient tissue for a full assay, single point assays were run. Treatment of Chickens. Baby chicks were purchased from a commercial hatchery and were fed a commercial growing mash during the entire experiment. The chickens were treated with the various stilbestrol preparations at the age of eight weeks, the implantations or injections being made subcutaneously at the base of the skull in each case. The birds were slaughtered 4 to 6 weeks after treatment. Immediately after slaughter, the feathers were removed, the necks severed near the shoulders, and the carcasses eviscerated. The carcasses and the various organs were placed in sealed polyethylene bags and sharp frozen. Feeding the chicken growing mash to immature mice for seven days did not result in any stimulation of uterine growth. Stilbestrol Preparations Studied. Four commercially available preparations for the stilbestrol treatment of poultry were selected based on the type of vehicle employed. The chickens received one treatment. The usual 12- or 15-mg. dose of stilbestrol was used in most cases, but in one experiment doses of one-half this amount were also used. The preparations used were as follows:
122
E. J. UMBERGER, G. H. GASS, K. J. DAVIS, J. M. CURTIS AND C. G. DURBIN T A B L E 3.—Experiment No. 2. Estrogenic residues the tissues of chickens 6 weeks after treatment with stilbestrol (DES) Treatment DES 12 15 15
Vehicle
III
in
Residual estrogen in DES Lean*
Liver
0.8 + 0.3 0.0 0.0
22.4+5.0 3.3±1.5 12.1±3.5
Gizzard Heart 0.0
—t 0.0 0.0
0.0=*Level of estrogenic activity not significantly different from controls. t=Significant estrogenic activity indicated but not quantitatively determined. * = Consisted of both leg and breast muscle, skin removed.
mg. liquid-rosin preparations, Cornish cockerels were used. Eighty-five chickens received the paste, 85 received the liquidrosin, and 350 were kept for the controls. They were treated at 8 weeks of age and slaughtered 6 weeks after treatment. Twenty-five eviscerated carcasses were saved from each treated group and each control group. In addition all livers, gizzards, and hearts were kept. The tissues were again fed to the mice raw. The results of the assays are shown in Table 3. As suspected, the'liver contained nearly all of the residual estrogen. Experiment No. 3. The chief purposes of this experiment were to determine whether the residual estrogenic activity found in the tissues persisted when the tissues were cooked and whether estrogenic residues are present in the skin. At the same time, other tissues and organs were examined for added estrogenic activity. Again the birds were treated at 8 weeks of age, but the treatment time was reduced to 4 weeks. The birds which received the 12-mg. pellet were Rhode Island Reds, those that received the 15-mg. liquid-rosin preparation were Cornish, and those that received the 15-mg. paste preparation were mixed meat breeds. There were approximately 125 treated birds and 375 control birds in each group. The results of the assays for residual estrogenic activity are shown in Table 4.
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the carcass lean meat of the chickens treated with the paste, liquid-rosin, or the liquid preparations, but that the flesh from the pellet-treated animals contained a slight amount as indicated by the bioassay. The significance of this result will be discussed later. No added estrogenic activity was detected in the offal or giblets of the chickens treated with the liquid preparation, but significant amounts were found in these tissues from chickens treated with the other three preparations. In most cases, responses were maximal. Since there was insufficient tissue remaining for further examination, a quantitative estimation of the amount of estrogen present was not possible. Approximately 18 heads from each treated group were examined grossly for implantation residues at the site of injection. No visible injection area was present from the paste or liquid preparations. Pellets were found in all cases, the average residue from the group treated with the 12-mg. pellet (15 mg. with excipient) being about 6.3 mg. With the liquid-rosin preparation, necrotic areas were found near the site of injection in most instances. The areas contained a yellow granular material similar to rosin, from which a qualitative test for stilbestrol was obtained. Experiment No. 2. This experiment was designed to confirm the results of Experiment No. 1. Particular attention was given to the liver since it was suspected that this was the tissue responsible for most of the estrogenic activity found in the giblets. Only the high dose levels of the pellet, paste, and liquid-rosin preparations were studied since no residues were found following the use of the liquid preparation. White Leghorn cockerels were used for the 12-mg. pellet study; 100 chickens received the pellet and 300 were kept as controls. For the 15-mg. paste and the 15-
ESTROGENIC RESIDUES IN TISSUE TABLE 4.—Experiment No. 3. Estrogenic residues in the tissues of chickens 4 weeks after treatment with stilbestrol (DES)
Tissues
Residual estrogen in DES equivalents (ppb.) 12 mg. pellet
15 mg. liquid-rosin
0.7±0.2 1.3 + 0.3 0.5+0.02 0.8 + 0.2 29.2 + 4.1 20.1±0.5 0.0 0.0 0.0 0.0
-t -t
0.0
35.3±8.5 MOO
-t
0.0 5.8+0.2 >100 >100
0.0=Level of estrogenic activity not significantly different from controls. t=Significant estrogenic activity indicated but not quantitatively determined. > =Level of estrogenic activity too great for calculation. Figure shown is a minimum value.
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All of the assays in this experiment were performed on cooked tissue. In order to follow a readily reproducible cooking method, the tissues^ after being ground and mixed with the laboratory diet, were cooked in an autoclave at 15 pounds' pressure for 20 minutes. Under these conditions the temperature attained is about 250°F. The diets were cooked in sealed stainless steel containers to prevent a change of weight. Before adding stilbestrol, the diets were thoroughly stirred with an ordinary electric kitchen mixer in order to have a uniform distribution of tissue and fluid. The results indicate that there was no destruction of the residual estogenic activity by cooking. When stilbestrol, estrone, or estradiol-17/3 were added to the diets before cooking and their effect on the uterine weight of mice compared with similar diets to which the estrogens were added after cooking, there was no difference in the uterine response, indicating no destruction of the estrogens by the cooking process. Because of the high fat content of chicken skin tissue, we were unable to assay satisfactorily this tissue for its estrogenic content. The mice do not readily consume a high fat content diet. In an-
other communication we have shown that the fat content of the diet alone will affect the uterine weight of immature mice, and that high fat diets result in a decreased response to added stilbestrol. By ether extraction we found that skin tissue from control chickens contained about 23 percent fat, and skin from the pellettreated chickens contained about 39 percent fat. To circumvent these difficulties, the fat was rendered from the skins by heating in an oven at 325°F. for two hours. The fat was then added to the regular laboratory mouse diet to the extent of 10 percent. Since the mice would consume only 2 to 3 grams of this diet daily as compared to 6 to 7 grams of the tissue diets, it was necessary to increase the amounts of stilbestrol added to both control and treated diets to 0, 6, 12, and 18 parts per billion. In this way we were able to show that skin fat contained 35 to over 100 parts per billion of stilbestrol equivalents of added estrogenic activity. Since gross examination of heads from the paste-treated chickens did not indicate any residue in the first experiment, heads were assayed by grinding and incorporating in the diet of mice in a rangefinder experiment. Maximum uterine stimulation was obtained at the lowest concentration of treated heads, indicating a large residue of stilbestrol at the implanation site. Similar results were obtained from the heads of birds treated with the liquid-rosin preparation. Experiment No. 4. Since we were able to find significant residual estrogenic activity in the livers of chickens treated under highly controlled experimental conditions, it seemed desirable to confirm our findings by showing that residues occurred in the livers of commercially treated chickens. Accordingly, liver from chickens having received no stilbestrol was obtained from a commercial plant in
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Lean, leg (dark meat) 1.8±0.2 Lean, breast (white meat) 1.0+0.3 Liver 25.6+6.6 Gizzard -t Hearts Kidney >8 Spleen 0.0 Offal 6.9±0.5 Skin Fat 36.5±9.8 Heads
15 mg. paste
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E. J. UMBERGER, G. H. GASS, K. J. DAVIS, J. M. CURTIS AND C. G. DURBIN
some of the uteri from animals on the highest dosage levels, slight endometrial edema and vascular congestion. The addition of more than 6 parts per billion to the control chicken tissue diets resulted in only slightly increased endometrial edema and vascular congestion. The uteri of mice fed diets prepared from livers or kidneys of stilbestroltreated chickens showed estrogenic activity without the addition of stilbestrol to the diets, and could not be distinguished morphologically from those of mice fed control diets with 6 parts per billion or more of added stilbestrol. On the other hand, microscopic morphological evidence of estrogenic stimulation was not seen in the uteri of mice fed lean meat tissue diets from control or treated chickens when no stilbestrol was added to the diets, or from high fat control diets. When judged by microscopic morphologic criteria, minimal uterine estrogenic response required the addition of at least 2 parts per billion of stilbestrol to the control tissue diets of the immature mice. DISCUSSION
Referring to Table 1, it is seen that several of the previous investigators employed assay methods using the vaginal smear as the criterion of response. Others, including ourselves, have employed methods based on an increase in the uterine weight. As pointed out by Rubin et al. (1951) the uterine weight method has the advantage of objectivity and is more sensitive. It is noted also that in only two of the ten studies shown in Table 1 were the tissues fed to the experimental animals. The other studies involved the extraction of the estrogen from the tissues and the subcutaneous injection of the extract or an oil solution of it. Lorenz (1954) criticized extraction methods on the basis of lack of
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Altanta, Georgia, and liver from chickens having received 12-mg. pellets was obtained in Portland, Maine. By using the former as a control tissue, the treated livers assayed 21.0 + 3.2 parts per billion stilbestrol equivalents. The diets prepared from the control liver from Atlanta caused no greater growth of the mouse uterus than did diets prepared from control liver obtained in our own experiments. The uniformity of response from control liver diets in the various experiments indicated that either natural estrogenic activity was absent or was not detectable by the assay method. Histological examination of the uteri of immature mice fed chicken tissue diets. Hooker (1945) has described in detail the morphological changes accompanying uterine stimulation by estrogens, androgens, and progestins in the castrate female mouse. Since uterine stimulation effecting an increase in weight may result from the administration of materials other than estrogens, the immature mouse uteri from several groups were examined microscopically for estrogenic stimulation. Miscroscopically, hematoxylin-eosin stained paraffin sections of the immature mouse uteri showed good morphological correlation with the stilbestrol levels in the diets fed the mice in these experiments. The uteri of immature mice fed diets of control chicken tissues showed small lumina, few glands, dense endometrial stroma, and inactive endometrial epithelium. The uteri of mice fed control chicken tissue diets with added stilbestrol in increasing amounts showed estrogenic effects correlating with the increased dosage levels. These effects consisted of larger uteri with larger lumina, more endometrial glands, and taller, more active epithelium, decreased density of the stroma, rounded stromal nuclei and, in
ESTROGENIC RESIDUES IN TISSUE
genic activity fround is due to stilbestrol and is being measured quantitatively. Our failure to find stilbestrol residues in the giblets or offal following treatment with the liquid preparation may be explained by the probability that the stilbestrol had disappeared from the injection site by the end of the five-week treatment period. It would appear that the level of estrogenic residues in the liver (or other tissue) is related to the amount of stilbestrol to which the bird is currently being exposed. This can be seen by comparing the amount of estrogen in the liver of chickens treated for four weeks (Table 4) with the amount present in the liver at the end of six weeks (Table 3). The fact that the concentration decreases would indicate that the storage is not accumulative. This may explain in part the failure of Bird et al. (1947), to detect stilbestrol in the abdominal fat of chickens treated with a total of 10 mg. of stilbestrol in oil in daily subcutaneous injections over a period of 12 days. On the other hand, in a publication from the same laboratory by Snair et al. (1954), it was stated that "residues containing significant amounts of stilbestrol have been recovered from the fatty tissue of poultry after the pellet implantation of this synthetic estrogen." Gowe (1949) pointed out that the ability to store estrogen may differ for abdominal fat and subcutaneous fat. Following the implantation of 48 mg. of stilbestrol in a turkey, the tissues were extracted and the extracts injected subcutaneously into the castrate female rat. Examination of the vaginal smears showed no sign of estrogenic stimulation from 1ml. doses of abdominal fat but strong stimulation from 1-ml. dose of skin fat. Greenblatt et al. (1952), also failed to detect estrogenic activity in an extract of fatty tissue from a pelleted chicken by us-
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proof of the efficiency of the various extraction procedures used. A further criticism of the methods based on the subcutaneous injection of tissue extracts is the difficulty of interpretation of the assay results. Lee, Robbins and Chen (1942) showed that the slope of the response curve for the naturally occurring estrogen estrone differed from that of stilbestrol in an assay method based on the uterine weight response of the immature female rat, and a similar conclusion was reached by Curtis, Umberger and Knudsen (1947) using the vaginal smear response of the adult ovariectomized rat. In their review of the literature on the relative potency of stilbestrol and estrone by the various assay methods, Lee, Robbins and Chen (1942) found that in spayed rats the ratio of activity of stilbestrol to estrone varied from 1 to 5 by subcutaneous administration and from 20 to 80 by oral administration. Since extraction methods would in all probability recover the naturally occurring estrogens as well as the stilbestrol, and since bioassays in which the response is a function of the log of the dose can be quantitatively interpreted only when the slope of the standard curve and the unknown curve are the same, the naturally occurring estrogens would most likely interfere with the determination of stilbestrol in subcutaneous administration methods. In the method used in the present work, the response (log of the ratio of the uterine weight to body weight) is a linear function of the dose for both the naturally occurring estrogens and the synthetic estrogen. Further, the naturally occurring estrogens are 1/10 to 1/20 as active as stilbestrol when added to the diet of the mouse. These observations, and the use of an experimental design in which tissues from control birds are compared directly with tissues from treated birds, increase the likelihood that the added estro-
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E. J. UMBERGER, G. H. GASS, K. J. DAVIS, J. M. CURTIS AND C. G. DURBIN
cerated carcases of chickens treated with a 12-mg. pellet as the treatment period was increased from one to four weeks. In the review by Lorenz (1954), it was pointed out that extra fat is deposited in all parts of the bird treated with estrogens. In a separate publication (Umberger and Gass, 1958) we have shown that the uterine weight of the immature mouse is dependent upon the fat content of the diet. Table 5, shows an analysis of the fat content of tissues assayed in Experiment No. 3. Careful analysis of our results would indicate that a difference in fat content of the tissues would not result in an overestimation of estrogenic residues of more than two parts per billion of stilbestrol equivalents. Histological examination of the uteri of mice fed diets prepared from leg or breast meat of the pelleted chickens failed to show any evidence of estrogenic stimulation. It is possible that a change in uterine weight is a more sensitive indicator of estrogenic activity than is histological examination of the uteri. I t is doubtful whether the small differences in fat content between control and treated leg meat tissues in the birds treated with the 15-mg. paste and the 15-mg. liquidrosin could account for the estrogenic activity found, but at the same time we are unable to say for certain whether the small amounts found are due to residual estrogen.
TABLE 5.—Fat content of chicken tissues measured as ether extractable solids and expressed as percent of fresh tissue (Experiment No. 3) 12 mg. pellet
15 mg. paste
15 mg. liquid-rosin
Tissue Lean, leg Lean, breast Liver Gizzards Intestines Skin
Control
Treated
Control
Treated
Control
Treated
2.54
4.33
3.70
6.10 8.67 39.0
4.57 1.43 5.21 7.93
3.33
5.49 2.52 22.8
4.53 0.81 3.54 7.48
7.54 23.7
10.55 30.0
24.6
32.3
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ing subcutaneous injection and the immature mouse uterine weight method. It is not clear, however, whether he used abdominal fat or subcutaneous fat, and the amount of extract is not specified. As pointed out earlier, we were unable to obtain sufficient abdominal fat from untreated birds for use in assaying the estrogen in the abdominal fat of treated birds. However, in Experiment No. 3 an assay was run in which the rendered abdominal fat from the 12-mg. pelleted birds was compared with rendered subcutaneous fat from control birds. There was no significant difference between the two responses, indicating an absence of estrogenic residues in the abdominal fat of pelleted birds. It will be noted that estrogenic residues of less than two parts per billion in stilbestrol equivalents were indicated by the bioassay method in the lean meat, both white and dark, of chickens slaughtered 4 weeks after treatment with the 12-mg. pellet, the 15-mg. paste, and the IS mg. liquid-rosin preparations (See Table 4). A decreasing amount was found in the lean meat of the pelleted birds at 5 and 6 weeks after treatment, but none was detectable at this time from the lean meat tissues of birds treated with the other two preparations. Stob, Andrews, Zarrow and Beeson (1954) also found a decrease in residual hormone activity in the evis-
ESTROGENIC RESIDUES IN TISSUE
SUMMARY
1. The edible tissues of chickens treated with stilbestrol in the form of a 12-mg. pellet, a 15-mg. paste, and a 15-mg. liquid-rosin preparation contain measurable amounts of added estrogenic activity due to the stilbestrol treatment four to five weeks after treatment. No estrogenic residues were detected five weeks after the use of a 15-mg. liquid preparation. 2. The amounts of such added estrogenic activity are equivalent to 20 to 30 parts per billion of stilbestrol in the liver and to 35 or more parts per billion in the fat of the skin. Detectable amounts occur also in the kidneys and in the small intestines of treated birds. 3. Small amounts of added estrogenic activity are indicated in the leg and breast lean meat of treated chickens. The amounts indicated are of the order of two parts per billion or less, the amount decreasing with the length of the treatment period. The possibility that these small amounts indicated by the bioassay may be the result of a difference in the fat content of the tissues of control and treated birds is discussed. 4. Cooking does not destroy the estro-
genic residues in the tissues. Also, cooking does not result in a loss of activity when stilbestrol, estrone, or estradiol-17(3 are added to the tissues before cooking. 5. The literature pertaining to the assay of edible tissues from stilbestroltreated poultry for stilbestrol residues has been reviewed. 6. Histological examination of the uteri of immature mice fed liver from treated chickens showed the same picture of estrogenic stimulation as those from mice fed a diet containing added stilbestrol. ACKNOWLEDGMENTS
The authors gratefully acknowledge the assistance of Paul C. Underwood, D.V.M., who treated and maintained the chickens, and to Leonard J. Davis for technical assistance in preparing and administering the diets to the mice. We also acknowledge the assistance of Robert E. Zwickey, D.V.M., who examined the chickens for their condition of health and for any possible conditions not associated with the estrogen treatment. REFERENCES Bird, S., L. I. Pugsiey and M. O. Klotz, 1947. The quantitative recovery of synthetic estrogens from tissues of birds (Gailus domeslicus), the response of the birds' testis, comb and epidermis to estrogen and of humans to ingestion of tissues from treated birds. Endocrinology, 41: 282-294. Campbell, E. D., and H. Wick, 1952. In U. S. Congress, House. Select Committee to Investigate the Use of Chemicals in Food and Cosmetics, 82nd Congress, 2nd Session. Hearings pursuant to H. Res. 74 and H. Res. 447. Part 3, Appendix B, pp. 1438-1439. Colombel, J., 1950. Toxicite des oestrogenes de synthase dans la chair des Coqs, castres chimiquement. Rec. de Med. Veterinaire Paris, 126: 100108. Curtis, J. M., E. J. Umberger and L. F. Knudsen, 1947. The interpretation of estrogenic assays. Endocrinology, 40: 231-240. Gowe, R. S., 1949. Residual estrogens in the tissues of fowl treated with dienestrol diacetate. Poultry Sci. 28: 666-669.
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In confirmation of the observations of Stob et al. (1954) in beef and Taylor and Gordon (1955) in swine, we have found that the added estrogenic activity demonstrated in stilbestrol-treated chickens is not destroyed by cooking. It was further demonstrated that when stilbestrol, estrone, or estradiol-17/3 is added to tissues, the cooking process does not result in a loss of estrogenic activity. Since the experiments reported here were performed on several different breeds of chickens, there appears to be no effect due to breed on the accumulation of estrogens in the edible tissues.
127
128
E. J. UMBEEGER, G. H. GASS, K. J. DAVIS, J. M. CURTIS AND C. G. DURBIN mouse uterine response. Endocrinology, 49: 429439. Snair, D. W., S. E. Jaffray, H. C. Grice and L. I. Pugsley, 1954. Studies on the subacute and chronic administration of stilbestrol in the male rat. Canadian J. Biochem. Physiol. 32: 41-49. Stob, M., F. N. Andrews and M. X . Zarrow, 1954. The detection of residual hormone in the meat of animals treated with synthetic estrogens. Am. J. Vet. Research, 15: 319-322. Stob, M., F. N. Andrews, M. X. Zarrow and W. M. Beeson, 1954. Estrogenic activity of the meat of cattle, sheep and poultry following treatment with synthetic estrogens and progesterone. J. Animal Sci. 13: 138-151. Swift, C. E., 1954. The diethylstilbestrol content of tissues of treated steers, lambs, and poultry. Food Research, 19: 402-409. Taylor, J. H., and W. S. Gordon, 1955. The effect of feeding a diet containing stilbestrol and thyroxine to growing pigs with special reference to the toxicity of stilbestrol. Vet. Record, 67: 48-52, 58. Umberger, E. J., G. H. Gass and J. M. Curtis, 1958. Design of a biological assay method for the detection and estimation of estrogenic residues in the edible tissues of domestic animals treated with estrogens. Endocrinology, 63: 806-815. Umberger, E. J., and G. H. Gass, 1958. The effect of dietary fat on the uterine weight response of immature mice to oral stilbestrol. Endocrinology, 63: 801-805.
Detection of Residual Estrogenic Activity in the Edible Tissues of Chickens Fed Dienestrol Diacetate ERNEST J. UMBERGER AND GEORGE H. GASS Food and Drug Administration, U. S. Department of Health, Education, and Welfare, Washington 25, D. C. (Received for publication May 23,1958)
T
HIS laboratory has recently completed a series of assays designed to show whether or not estrogenic residues are present in the edible tissues of steers fed stilbestrol (Umberger, Curtis and Gass, 1959) or in the edible tissues of chickens injected with various preparations of stilbestrol (Umberger, Gass, Davis, Curtis and Durbin, 1959) to pro-
mote growth and fattening. For this purpose an assay method was developed based on the oral feeding of the tissue to the immature mouse and the use of the uterine weight as the criterion of response (Umberger, Gass and Curtis, 1958). An alternative method for estrogenizing poultry is the addition of dienestrol
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Greenblatt, R. B., and N. H. Brown, 19S2. The storage of estrogen in human fat after estrogen administration. Am. J. Gynec. Obstet. 61: 13611363. Hooker, C. W., 1945. A criterion of luteal activity in the mouse. Anatomical Record, 93: 333-347. Jones, O., and F. E. Deatherage, 1953. The diethylstilbestrol content of the meat from chickens treated with this estrogenic substance. Food Research, 18: 30-34. Koch, W., and G. Heim, 1952. Die Speicherung von Oestrogenen im Tierkorper. I. Hormonal kastrierte Hahnchen. Endokrinologie, 29: 288-293. Lee, H. M., E. B. Robbins and K. K. Chen, 1942. The potency of stilbestrol in the immature female rat. Endocrinology, 30: 469-473. Lorenz, F. W., 1954. Effects of estrogens on domestic fowl and applications in the poultry industry. Vitamins and Hormones, 12: 235-275. Merker, P. C , L. D. Edwards, F. N. Andrews and J. E. Christian, 1955. The estrogenic activity of residues obtained from chickens treated with pellets of diethylstilbestrol. Poultry Sci. 34:11181125. Meyer, R. K., 1952. Endocrine Laboratories Report No. 1EL-270. In U. S. Congress, House. Select Committee to Investigate the Use of Chemicals in Food and Cosmetics, 82nd Congress, 2nd Session. Hearings pursuant to H. Res. 74 and H. Res. 447. Part 3, Appendix B, pp. 1436-1438. Rubin, B. L., A. S. Dorfman, L. Black and R. I. Dorfman, 1951. Bioassay of estrogens using the
1
P. B, SlEGEL
CONCLUSIONS
Significant differences among lines of White Plymouth Rock cocks were found for aggressiveness and sex drive, indicating a genetic basis for these traits. No heterotic effect, with regard to these characteristics, was exhibited when cross line cocks were compared with cocks from the
inbred lines from which they originated. Correlations between the behavior traits studied indicate a positive relationship between aggressiveness and mating ability. Correlations among the 4 types of mating behavior measured, indicate that males which exhibit the largest number of courts, mounts, and treads, also complete the largest number of matings. REFERENCES Guhl, A. M., 1953. Social behavior of the domestic fowl. Kansas Agr. Expt. Sta. Tech. Bui. 73. Guhl, A. M., 1956. Personal communication. Guhl, A. M., N. E. Collias and W. C. Allee, 1945. Mating behavior and the social hierarchy in small flocks of White Leghorns. Physiol. Zool. 18: 365-390. Lerner, I. M., 1954. Genetic Homeostasis. John Wiley & Sons, Inc. New York. Valenstein, E. S., W. Riss and W. C. Young, 1954. Sex drive in genetically heterogeneous and highly inbred strains of male guinea pigs. J. Comp. Physiol. Psychol. 47: 162-165. Wood-Gush, D. G. M., 1956. The agonistic and courtship behavior in the Brown Leghorn cock. Brit. J. An. Behav. 4: 133-142. Wood-Gush, D. G. M., 1957. Aggression and sexual activity in the Brown Leghorn cock. Brit. J. An. Behav. 5: 1-6. Wood-Gush, D. G. M., and R. Osborne, 1956. A study of differences in the sex drive of cocks. Brit. J. An. Behav. 4: 102-110.
Estrogenic Residues in the Edible Tissues of Stilbestrol-Fattened Chickens ERNEST J. UMBERGEE, GEORGE H. GASS, KENT J. DAVIS, JACK M. CURTIS AND CHARLES G. DURBIN
Food and Drug Administration, U. S. Department of Health, Education, and Welfare, Washington, D. C. (Received for publication May 23, 1958)
T
HE use of synthetic estrogens for the hormonal fattening of poultry, and studies designed to demonstrate whether or not estrogenic residues remain in the
edible tissues of the treated poultry have recently been reviewed by Lorenz (1954) and by Merker et al. (1955). Commercial use of the estrogens has been limited to the
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not always lower than that of the heterogeneous stocks. The significance of pen effects points out that experimental design is important in testing for sex drive. Pen effects were much larger than had been anticipated. If all males had not been tested with all females, much of the information obtained in this study would have been invalid. The barely significant correlations between encounters won with number of courts and completed matings support the thesis of Wood-Gush (1956) that there may be a relationship between aggressive elements and courtship displays. However, the non-significant correlation between aggressiveness and both mounts and threads indicates that this relationship is probably minor. The highly significant correlations between the four types of mating behavior measured show a high degree of relationship among them.
ESTROGENIC RESIDUES IN TISSUE
EXPERIMENTAL
Assay Methods. The details of the bioassay method and the experimental de-
signs are described in a paper by Umberger, Gass and Curtis (1958). In general, the method consisted of grinding 100 parts of tissue with 10 to 20 parts of laboratory diet and feeding these diets to immature female mice for seven days. On the eighth day the animals were sacrificed and their uteri weighed. Each animal received 6 or 7 grams of diet per day, an amount which was fully consumed and permitted some growth. Usually, six mice at each dosage level were used. To obtain a quantitative measurement of added estrogenic activity in the treated tissues, and at the same time to estimate the sensitivity of the method, three experimental designs were used. In the first design, stilbestrol was added to both control and treated tissue diets fed to the mice at levels of 0, 2, 4, and 6 parts per billion. At these very low dosage levels, a linear relationship exists when the dose is plotted versus the log of the ratio of the uterine weight in milligrams to the final body weight in grams. Any displacement of the treated tissue curve away from the control curve along the response axis measures the amount of estrogenic residues in equivalents of stilbestrol. When the level of estrogenic activity was greater than 2 parts per billion, a second design termed "the rangefinder" was used. This consisted of feeding control tissue diets, to which had been added 0, 3, and 6 parts per billion of stilbestrol, to three groups of mice and at the same time feeding treated tissue diets, diluted with 0, 50, 75, 87.5, and 93.75 percent control tissue diet, to five other groups of mice. By proper selection of responses, the data could be calculated as a balanced slope-ratio assay, or the data could be used to estimate graphically a dilution factor for use in the third experimental design. In this third design the treated tissue diet was diluted with control tissue diet so that the expected
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t
implantation or injection of stilbestrol and to the oral feeding of dienestrol diacetate. The usual practice is to treat the birds at 7 to 9 weeks of age and to slaughter the birds 4 to 6 week later. With the development of a sensitive assay method (Umberger, Gass and Curtis, 1958) based on the work of Stob, Andrews and Zarrow (1954), an examination has been made of the edible tissues from chickens treated with various commercial preparations of stilbestrol. The results of these assays are the subject of this report. A subsequent report will detail the studies with dienestrol diacetate. The literature contains references to both biological and chemical assay methods for the detection and estimation of estrogenic residues in tissues of poultry following stilbestrol treatment. Table 1 summarizes in chronological order all the references available to us using biological assay methods. It will be noted that data are available on both chickens and turkeys, and that with one exception the method of treatment was by pellet implantation. We note also that in only two of the ten cases were the tissues fed to the assay animals; the other assays involved solvent extraction of the tissues and subcutaneous injection of the extract dissolved in oil. Studies involving chemical assay for stilbestrol in tissues were those of Jones and Deatherage (1953) and Swift (1954). The former were unable to detect any estrogen in edible tissues, but did find residues at the site of injection of a 12-mg. pellet or a 15-mg. paste preparation. Swift (1954) found evidence of small amounts of stilbestrol in extracts of liver and skin, but not in the muscle of cockerels treated with a 15-mg. pellet of stilbestrol.
119
12 mg. pellet
Chicken
Turkey
Campbell et al. (1952)
Greenblatt et al. (1952)
Koch et al. (1952)
Stob et al. (1954)
Snair et al. (1954)
Merker et al. (1955)
5
6
7
8
9
10
12 mg, pellet
12 mg. pellet
12 mg. pellet
20 mg. pellet
15 mg. pellet 30 mg. pellet
12 mg. pellet
Carcass
Fat
Carcass
Muscle Bones Skin Liver and gall Abd. fat
Fat
Carcass Liver Liver
Carcass Liver
Liver
* DE—Stilbestrol equivalents. f EE—Estrone equivalents. X Authors also calculate in DE using factor 0.08/0.06.
Chicken
Chicken
Chicken
Chicken
Chicken
12 mg. pellet
Chicken
Meyer (1952)
4
15 mg. pellet 30 mg. pellet
Chicken
Colombel (1950)
3
Abd. Fat Liver Fat Skin-muscle
48 mg. pellet
Turkey
Gowe (1949)
2
Abdominal Eat
10 mg. in oil
Stilbestrol preparation
Chicken
Species
Bird et al. (1947)
Investigators
1
No.
Tissue assayed
Vaginal Smear Vaginal Smear
Mouse Castrate Mouse Castrate
Ext.-chloroform Ext.-chloroform
s.c. oil
Oral s.c. s.c. oil
Uterine Weight Vaginal Smear
Uterine Weight Vaginal Smear Uterine Weight
Mouse Immature Mouse Castrate
Mouse Castrate Rat Castrate Mouse Castrate
Ext.-ether
In food Rendered Ext.-ethanol, ether, alkali, ether
s.c. oil
s.c. oil
s.c. oil
Oral
s.c. oil
s.c. oil
Route
Ext.-benzene-ethanol
Vaginal Smear
Rat Castrate
In food
Vaginal Smear
Vaginal Smear
Response
Rat Castrate
Rat Castrate
Animal
Method of assay
Ext.-methanol, ether
Ext.-ether
Preparation of sample Results
Absent
Present
Present
Present in all. From 0.01 to 34 mg. per 2,200 g. bird
Absent
Absentf 60-118 ppb. E E 50 ppb. E E
13+ 3 p p b . E E f 24±19ppb. EE
210 jug. per 35 g. liver (6,000 ppb. DE*)
Absent Present Present
Absent
of the literature on the biological assay of the edible tissues of stilbestrol-treated poultry for estrogenic residues
Treatment
TABLE 1.--Summary
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ESTROGENIC RESIDUES IN TISSUE
Pellet: A 15-mg. pellet containing 12 mg. of stilbestrol. Paste: A preparation of paste-like consistency having a water-soluble base and a wax-like substance to delay absorption of the stilbestrol. Liquid-Rosin: A preparation of liquid
consistency having a water-soluble base and gum rosin to delay absorption. Liquid: A preparation of liquid consistency having only a water-soluble base. Experiment No. 1. 400 New Hampshire cockerels were divided into 16 groups of 25 each. Eight of these groups were kept for controls; the other 8 groups were treated with the four preparations containing two dosage levels of stilbestrol. The birds were treated at 8 weeks of age and were slaughtered 5 weeks after treatment. The following tissues and organs were saved: Eviscerated carcass, giblets (heart, gizzard, and liver), offal (small intestines, split and washed), and heads (from treated groups only). Abdominal adipose tissue was not saved since the only group which had any appreciable amount was the pelleted group, and therefore, sufficient control tissue for an assay was unavailable. Tissue assays were made on lean meat trimmed from the eviscerated carcasses, on the giblets, and on the offal. The results are shown in Table 2. In this experiment the tissues were fed raw. It will be noted that no estrogenic residues were found in T A B L E 2.—Experiment No. 1. Estrogenic residues the tissues of chickens 5 weeks after treatment with stilbestrol (DES) Treatment
in
Residual estrogen in DES equivalents (ppb.)
mg. DES
Vehicle
Lean*
Giblets
Offal
12 15 15 15 6 7.5 7.5 7.5
Pellet Paste Liquid-Rosin Liquid Pellet Paste Liquid-Rosin Liquid
0.8±0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0
>6 >6 >4 0.0 >6 2.0
>5 >5 0.8+0.4 0.0
-t
0.0
0.0=Level of estrogenic activity not significantly different from controls. t=Significant estrogenic activity indicated but not quantitatively determined. > =Level of estrogenic activity too great for calculation. Figure shown is a minimum value. * = Consisted of both leg and breast muscle, skin removed.
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•
concentration of estrogenic activity in stilbestrol equivalents was between 1 and 2 parts per billion. The control tissue diet and the diluted treated tissue diet were then spiked with 0, 2, 4, and 6 parts per billion of stilbestrol as in the first experimental design. When there was insufficient tissue for a full assay, single point assays were run. Treatment of Chickens. Baby chicks were purchased from a commercial hatchery and were fed a commercial growing mash during the entire experiment. The chickens were treated with the various stilbestrol preparations at the age of eight weeks, the implantations or injections being made subcutaneously at the base of the skull in each case. The birds were slaughtered 4 to 6 weeks after treatment. Immediately after slaughter, the feathers were removed, the necks severed near the shoulders, and the carcasses eviscerated. The carcasses and the various organs were placed in sealed polyethylene bags and sharp frozen. Feeding the chicken growing mash to immature mice for seven days did not result in any stimulation of uterine growth. Stilbestrol Preparations Studied. Four commercially available preparations for the stilbestrol treatment of poultry were selected based on the type of vehicle employed. The chickens received one treatment. The usual 12- or 15-mg. dose of stilbestrol was used in most cases, but in one experiment doses of one-half this amount were also used. The preparations used were as follows:
122
E. J. UMBERGER, G. H. GASS, K. J. DAVIS, J. M. CURTIS AND C. G. DURBIN T A B L E 3.—Experiment No. 2. Estrogenic residues the tissues of chickens 6 weeks after treatment with stilbestrol (DES) Treatment DES 12 15 15
Vehicle
III
in
Residual estrogen in DES Lean*
Liver
0.8 + 0.3 0.0 0.0
22.4+5.0 3.3±1.5 12.1±3.5
Gizzard Heart 0.0
—t 0.0 0.0
0.0=*Level of estrogenic activity not significantly different from controls. t=Significant estrogenic activity indicated but not quantitatively determined. * = Consisted of both leg and breast muscle, skin removed.
mg. liquid-rosin preparations, Cornish cockerels were used. Eighty-five chickens received the paste, 85 received the liquidrosin, and 350 were kept for the controls. They were treated at 8 weeks of age and slaughtered 6 weeks after treatment. Twenty-five eviscerated carcasses were saved from each treated group and each control group. In addition all livers, gizzards, and hearts were kept. The tissues were again fed to the mice raw. The results of the assays are shown in Table 3. As suspected, the'liver contained nearly all of the residual estrogen. Experiment No. 3. The chief purposes of this experiment were to determine whether the residual estrogenic activity found in the tissues persisted when the tissues were cooked and whether estrogenic residues are present in the skin. At the same time, other tissues and organs were examined for added estrogenic activity. Again the birds were treated at 8 weeks of age, but the treatment time was reduced to 4 weeks. The birds which received the 12-mg. pellet were Rhode Island Reds, those that received the 15-mg. liquid-rosin preparation were Cornish, and those that received the 15-mg. paste preparation were mixed meat breeds. There were approximately 125 treated birds and 375 control birds in each group. The results of the assays for residual estrogenic activity are shown in Table 4.
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the carcass lean meat of the chickens treated with the paste, liquid-rosin, or the liquid preparations, but that the flesh from the pellet-treated animals contained a slight amount as indicated by the bioassay. The significance of this result will be discussed later. No added estrogenic activity was detected in the offal or giblets of the chickens treated with the liquid preparation, but significant amounts were found in these tissues from chickens treated with the other three preparations. In most cases, responses were maximal. Since there was insufficient tissue remaining for further examination, a quantitative estimation of the amount of estrogen present was not possible. Approximately 18 heads from each treated group were examined grossly for implantation residues at the site of injection. No visible injection area was present from the paste or liquid preparations. Pellets were found in all cases, the average residue from the group treated with the 12-mg. pellet (15 mg. with excipient) being about 6.3 mg. With the liquid-rosin preparation, necrotic areas were found near the site of injection in most instances. The areas contained a yellow granular material similar to rosin, from which a qualitative test for stilbestrol was obtained. Experiment No. 2. This experiment was designed to confirm the results of Experiment No. 1. Particular attention was given to the liver since it was suspected that this was the tissue responsible for most of the estrogenic activity found in the giblets. Only the high dose levels of the pellet, paste, and liquid-rosin preparations were studied since no residues were found following the use of the liquid preparation. White Leghorn cockerels were used for the 12-mg. pellet study; 100 chickens received the pellet and 300 were kept as controls. For the 15-mg. paste and the 15-
ESTROGENIC RESIDUES IN TISSUE TABLE 4.—Experiment No. 3. Estrogenic residues in the tissues of chickens 4 weeks after treatment with stilbestrol (DES)
Tissues
Residual estrogen in DES equivalents (ppb.) 12 mg. pellet
15 mg. liquid-rosin
0.7±0.2 1.3 + 0.3 0.5+0.02 0.8 + 0.2 29.2 + 4.1 20.1±0.5 0.0 0.0 0.0 0.0
-t -t
0.0
35.3±8.5 MOO
-t
0.0 5.8+0.2 >100 >100
0.0=Level of estrogenic activity not significantly different from controls. t=Significant estrogenic activity indicated but not quantitatively determined. > =Level of estrogenic activity too great for calculation. Figure shown is a minimum value.
|
All of the assays in this experiment were performed on cooked tissue. In order to follow a readily reproducible cooking method, the tissues^ after being ground and mixed with the laboratory diet, were cooked in an autoclave at 15 pounds' pressure for 20 minutes. Under these conditions the temperature attained is about 250°F. The diets were cooked in sealed stainless steel containers to prevent a change of weight. Before adding stilbestrol, the diets were thoroughly stirred with an ordinary electric kitchen mixer in order to have a uniform distribution of tissue and fluid. The results indicate that there was no destruction of the residual estogenic activity by cooking. When stilbestrol, estrone, or estradiol-17/3 were added to the diets before cooking and their effect on the uterine weight of mice compared with similar diets to which the estrogens were added after cooking, there was no difference in the uterine response, indicating no destruction of the estrogens by the cooking process. Because of the high fat content of chicken skin tissue, we were unable to assay satisfactorily this tissue for its estrogenic content. The mice do not readily consume a high fat content diet. In an-
other communication we have shown that the fat content of the diet alone will affect the uterine weight of immature mice, and that high fat diets result in a decreased response to added stilbestrol. By ether extraction we found that skin tissue from control chickens contained about 23 percent fat, and skin from the pellettreated chickens contained about 39 percent fat. To circumvent these difficulties, the fat was rendered from the skins by heating in an oven at 325°F. for two hours. The fat was then added to the regular laboratory mouse diet to the extent of 10 percent. Since the mice would consume only 2 to 3 grams of this diet daily as compared to 6 to 7 grams of the tissue diets, it was necessary to increase the amounts of stilbestrol added to both control and treated diets to 0, 6, 12, and 18 parts per billion. In this way we were able to show that skin fat contained 35 to over 100 parts per billion of stilbestrol equivalents of added estrogenic activity. Since gross examination of heads from the paste-treated chickens did not indicate any residue in the first experiment, heads were assayed by grinding and incorporating in the diet of mice in a rangefinder experiment. Maximum uterine stimulation was obtained at the lowest concentration of treated heads, indicating a large residue of stilbestrol at the implanation site. Similar results were obtained from the heads of birds treated with the liquid-rosin preparation. Experiment No. 4. Since we were able to find significant residual estrogenic activity in the livers of chickens treated under highly controlled experimental conditions, it seemed desirable to confirm our findings by showing that residues occurred in the livers of commercially treated chickens. Accordingly, liver from chickens having received no stilbestrol was obtained from a commercial plant in
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Lean, leg (dark meat) 1.8±0.2 Lean, breast (white meat) 1.0+0.3 Liver 25.6+6.6 Gizzard -t Hearts Kidney >8 Spleen 0.0 Offal 6.9±0.5 Skin Fat 36.5±9.8 Heads
15 mg. paste
123
124
E. J. UMBERGER, G. H. GASS, K. J. DAVIS, J. M. CURTIS AND C. G. DURBIN
some of the uteri from animals on the highest dosage levels, slight endometrial edema and vascular congestion. The addition of more than 6 parts per billion to the control chicken tissue diets resulted in only slightly increased endometrial edema and vascular congestion. The uteri of mice fed diets prepared from livers or kidneys of stilbestroltreated chickens showed estrogenic activity without the addition of stilbestrol to the diets, and could not be distinguished morphologically from those of mice fed control diets with 6 parts per billion or more of added stilbestrol. On the other hand, microscopic morphological evidence of estrogenic stimulation was not seen in the uteri of mice fed lean meat tissue diets from control or treated chickens when no stilbestrol was added to the diets, or from high fat control diets. When judged by microscopic morphologic criteria, minimal uterine estrogenic response required the addition of at least 2 parts per billion of stilbestrol to the control tissue diets of the immature mice. DISCUSSION
Referring to Table 1, it is seen that several of the previous investigators employed assay methods using the vaginal smear as the criterion of response. Others, including ourselves, have employed methods based on an increase in the uterine weight. As pointed out by Rubin et al. (1951) the uterine weight method has the advantage of objectivity and is more sensitive. It is noted also that in only two of the ten studies shown in Table 1 were the tissues fed to the experimental animals. The other studies involved the extraction of the estrogen from the tissues and the subcutaneous injection of the extract or an oil solution of it. Lorenz (1954) criticized extraction methods on the basis of lack of
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Altanta, Georgia, and liver from chickens having received 12-mg. pellets was obtained in Portland, Maine. By using the former as a control tissue, the treated livers assayed 21.0 + 3.2 parts per billion stilbestrol equivalents. The diets prepared from the control liver from Atlanta caused no greater growth of the mouse uterus than did diets prepared from control liver obtained in our own experiments. The uniformity of response from control liver diets in the various experiments indicated that either natural estrogenic activity was absent or was not detectable by the assay method. Histological examination of the uteri of immature mice fed chicken tissue diets. Hooker (1945) has described in detail the morphological changes accompanying uterine stimulation by estrogens, androgens, and progestins in the castrate female mouse. Since uterine stimulation effecting an increase in weight may result from the administration of materials other than estrogens, the immature mouse uteri from several groups were examined microscopically for estrogenic stimulation. Miscroscopically, hematoxylin-eosin stained paraffin sections of the immature mouse uteri showed good morphological correlation with the stilbestrol levels in the diets fed the mice in these experiments. The uteri of immature mice fed diets of control chicken tissues showed small lumina, few glands, dense endometrial stroma, and inactive endometrial epithelium. The uteri of mice fed control chicken tissue diets with added stilbestrol in increasing amounts showed estrogenic effects correlating with the increased dosage levels. These effects consisted of larger uteri with larger lumina, more endometrial glands, and taller, more active epithelium, decreased density of the stroma, rounded stromal nuclei and, in
ESTROGENIC RESIDUES IN TISSUE
genic activity fround is due to stilbestrol and is being measured quantitatively. Our failure to find stilbestrol residues in the giblets or offal following treatment with the liquid preparation may be explained by the probability that the stilbestrol had disappeared from the injection site by the end of the five-week treatment period. It would appear that the level of estrogenic residues in the liver (or other tissue) is related to the amount of stilbestrol to which the bird is currently being exposed. This can be seen by comparing the amount of estrogen in the liver of chickens treated for four weeks (Table 4) with the amount present in the liver at the end of six weeks (Table 3). The fact that the concentration decreases would indicate that the storage is not accumulative. This may explain in part the failure of Bird et al. (1947), to detect stilbestrol in the abdominal fat of chickens treated with a total of 10 mg. of stilbestrol in oil in daily subcutaneous injections over a period of 12 days. On the other hand, in a publication from the same laboratory by Snair et al. (1954), it was stated that "residues containing significant amounts of stilbestrol have been recovered from the fatty tissue of poultry after the pellet implantation of this synthetic estrogen." Gowe (1949) pointed out that the ability to store estrogen may differ for abdominal fat and subcutaneous fat. Following the implantation of 48 mg. of stilbestrol in a turkey, the tissues were extracted and the extracts injected subcutaneously into the castrate female rat. Examination of the vaginal smears showed no sign of estrogenic stimulation from 1ml. doses of abdominal fat but strong stimulation from 1-ml. dose of skin fat. Greenblatt et al. (1952), also failed to detect estrogenic activity in an extract of fatty tissue from a pelleted chicken by us-
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proof of the efficiency of the various extraction procedures used. A further criticism of the methods based on the subcutaneous injection of tissue extracts is the difficulty of interpretation of the assay results. Lee, Robbins and Chen (1942) showed that the slope of the response curve for the naturally occurring estrogen estrone differed from that of stilbestrol in an assay method based on the uterine weight response of the immature female rat, and a similar conclusion was reached by Curtis, Umberger and Knudsen (1947) using the vaginal smear response of the adult ovariectomized rat. In their review of the literature on the relative potency of stilbestrol and estrone by the various assay methods, Lee, Robbins and Chen (1942) found that in spayed rats the ratio of activity of stilbestrol to estrone varied from 1 to 5 by subcutaneous administration and from 20 to 80 by oral administration. Since extraction methods would in all probability recover the naturally occurring estrogens as well as the stilbestrol, and since bioassays in which the response is a function of the log of the dose can be quantitatively interpreted only when the slope of the standard curve and the unknown curve are the same, the naturally occurring estrogens would most likely interfere with the determination of stilbestrol in subcutaneous administration methods. In the method used in the present work, the response (log of the ratio of the uterine weight to body weight) is a linear function of the dose for both the naturally occurring estrogens and the synthetic estrogen. Further, the naturally occurring estrogens are 1/10 to 1/20 as active as stilbestrol when added to the diet of the mouse. These observations, and the use of an experimental design in which tissues from control birds are compared directly with tissues from treated birds, increase the likelihood that the added estro-
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cerated carcases of chickens treated with a 12-mg. pellet as the treatment period was increased from one to four weeks. In the review by Lorenz (1954), it was pointed out that extra fat is deposited in all parts of the bird treated with estrogens. In a separate publication (Umberger and Gass, 1958) we have shown that the uterine weight of the immature mouse is dependent upon the fat content of the diet. Table 5, shows an analysis of the fat content of tissues assayed in Experiment No. 3. Careful analysis of our results would indicate that a difference in fat content of the tissues would not result in an overestimation of estrogenic residues of more than two parts per billion of stilbestrol equivalents. Histological examination of the uteri of mice fed diets prepared from leg or breast meat of the pelleted chickens failed to show any evidence of estrogenic stimulation. It is possible that a change in uterine weight is a more sensitive indicator of estrogenic activity than is histological examination of the uteri. I t is doubtful whether the small differences in fat content between control and treated leg meat tissues in the birds treated with the 15-mg. paste and the 15-mg. liquidrosin could account for the estrogenic activity found, but at the same time we are unable to say for certain whether the small amounts found are due to residual estrogen.
TABLE 5.—Fat content of chicken tissues measured as ether extractable solids and expressed as percent of fresh tissue (Experiment No. 3) 12 mg. pellet
15 mg. paste
15 mg. liquid-rosin
Tissue Lean, leg Lean, breast Liver Gizzards Intestines Skin
Control
Treated
Control
Treated
Control
Treated
2.54
4.33
3.70
6.10 8.67 39.0
4.57 1.43 5.21 7.93
3.33
5.49 2.52 22.8
4.53 0.81 3.54 7.48
7.54 23.7
10.55 30.0
24.6
32.3
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ing subcutaneous injection and the immature mouse uterine weight method. It is not clear, however, whether he used abdominal fat or subcutaneous fat, and the amount of extract is not specified. As pointed out earlier, we were unable to obtain sufficient abdominal fat from untreated birds for use in assaying the estrogen in the abdominal fat of treated birds. However, in Experiment No. 3 an assay was run in which the rendered abdominal fat from the 12-mg. pelleted birds was compared with rendered subcutaneous fat from control birds. There was no significant difference between the two responses, indicating an absence of estrogenic residues in the abdominal fat of pelleted birds. It will be noted that estrogenic residues of less than two parts per billion in stilbestrol equivalents were indicated by the bioassay method in the lean meat, both white and dark, of chickens slaughtered 4 weeks after treatment with the 12-mg. pellet, the 15-mg. paste, and the IS mg. liquid-rosin preparations (See Table 4). A decreasing amount was found in the lean meat of the pelleted birds at 5 and 6 weeks after treatment, but none was detectable at this time from the lean meat tissues of birds treated with the other two preparations. Stob, Andrews, Zarrow and Beeson (1954) also found a decrease in residual hormone activity in the evis-
ESTROGENIC RESIDUES IN TISSUE
SUMMARY
1. The edible tissues of chickens treated with stilbestrol in the form of a 12-mg. pellet, a 15-mg. paste, and a 15-mg. liquid-rosin preparation contain measurable amounts of added estrogenic activity due to the stilbestrol treatment four to five weeks after treatment. No estrogenic residues were detected five weeks after the use of a 15-mg. liquid preparation. 2. The amounts of such added estrogenic activity are equivalent to 20 to 30 parts per billion of stilbestrol in the liver and to 35 or more parts per billion in the fat of the skin. Detectable amounts occur also in the kidneys and in the small intestines of treated birds. 3. Small amounts of added estrogenic activity are indicated in the leg and breast lean meat of treated chickens. The amounts indicated are of the order of two parts per billion or less, the amount decreasing with the length of the treatment period. The possibility that these small amounts indicated by the bioassay may be the result of a difference in the fat content of the tissues of control and treated birds is discussed. 4. Cooking does not destroy the estro-
genic residues in the tissues. Also, cooking does not result in a loss of activity when stilbestrol, estrone, or estradiol-17(3 are added to the tissues before cooking. 5. The literature pertaining to the assay of edible tissues from stilbestroltreated poultry for stilbestrol residues has been reviewed. 6. Histological examination of the uteri of immature mice fed liver from treated chickens showed the same picture of estrogenic stimulation as those from mice fed a diet containing added stilbestrol. ACKNOWLEDGMENTS
The authors gratefully acknowledge the assistance of Paul C. Underwood, D.V.M., who treated and maintained the chickens, and to Leonard J. Davis for technical assistance in preparing and administering the diets to the mice. We also acknowledge the assistance of Robert E. Zwickey, D.V.M., who examined the chickens for their condition of health and for any possible conditions not associated with the estrogen treatment. REFERENCES Bird, S., L. I. Pugsiey and M. O. Klotz, 1947. The quantitative recovery of synthetic estrogens from tissues of birds (Gailus domeslicus), the response of the birds' testis, comb and epidermis to estrogen and of humans to ingestion of tissues from treated birds. Endocrinology, 41: 282-294. Campbell, E. D., and H. Wick, 1952. In U. S. Congress, House. Select Committee to Investigate the Use of Chemicals in Food and Cosmetics, 82nd Congress, 2nd Session. Hearings pursuant to H. Res. 74 and H. Res. 447. Part 3, Appendix B, pp. 1438-1439. Colombel, J., 1950. Toxicite des oestrogenes de synthase dans la chair des Coqs, castres chimiquement. Rec. de Med. Veterinaire Paris, 126: 100108. Curtis, J. M., E. J. Umberger and L. F. Knudsen, 1947. The interpretation of estrogenic assays. Endocrinology, 40: 231-240. Gowe, R. S., 1949. Residual estrogens in the tissues of fowl treated with dienestrol diacetate. Poultry Sci. 28: 666-669.
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In confirmation of the observations of Stob et al. (1954) in beef and Taylor and Gordon (1955) in swine, we have found that the added estrogenic activity demonstrated in stilbestrol-treated chickens is not destroyed by cooking. It was further demonstrated that when stilbestrol, estrone, or estradiol-17/3 is added to tissues, the cooking process does not result in a loss of estrogenic activity. Since the experiments reported here were performed on several different breeds of chickens, there appears to be no effect due to breed on the accumulation of estrogens in the edible tissues.
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E. J. UMBEEGER, G. H. GASS, K. J. DAVIS, J. M. CURTIS AND C. G. DURBIN mouse uterine response. Endocrinology, 49: 429439. Snair, D. W., S. E. Jaffray, H. C. Grice and L. I. Pugsley, 1954. Studies on the subacute and chronic administration of stilbestrol in the male rat. Canadian J. Biochem. Physiol. 32: 41-49. Stob, M., F. N. Andrews and M. X . Zarrow, 1954. The detection of residual hormone in the meat of animals treated with synthetic estrogens. Am. J. Vet. Research, 15: 319-322. Stob, M., F. N. Andrews, M. X. Zarrow and W. M. Beeson, 1954. Estrogenic activity of the meat of cattle, sheep and poultry following treatment with synthetic estrogens and progesterone. J. Animal Sci. 13: 138-151. Swift, C. E., 1954. The diethylstilbestrol content of tissues of treated steers, lambs, and poultry. Food Research, 19: 402-409. Taylor, J. H., and W. S. Gordon, 1955. The effect of feeding a diet containing stilbestrol and thyroxine to growing pigs with special reference to the toxicity of stilbestrol. Vet. Record, 67: 48-52, 58. Umberger, E. J., G. H. Gass and J. M. Curtis, 1958. Design of a biological assay method for the detection and estimation of estrogenic residues in the edible tissues of domestic animals treated with estrogens. Endocrinology, 63: 806-815. Umberger, E. J., and G. H. Gass, 1958. The effect of dietary fat on the uterine weight response of immature mice to oral stilbestrol. Endocrinology, 63: 801-805.
Detection of Residual Estrogenic Activity in the Edible Tissues of Chickens Fed Dienestrol Diacetate ERNEST J. UMBERGER AND GEORGE H. GASS Food and Drug Administration, U. S. Department of Health, Education, and Welfare, Washington 25, D. C. (Received for publication May 23,1958)
T
HIS laboratory has recently completed a series of assays designed to show whether or not estrogenic residues are present in the edible tissues of steers fed stilbestrol (Umberger, Curtis and Gass, 1959) or in the edible tissues of chickens injected with various preparations of stilbestrol (Umberger, Gass, Davis, Curtis and Durbin, 1959) to pro-
mote growth and fattening. For this purpose an assay method was developed based on the oral feeding of the tissue to the immature mouse and the use of the uterine weight as the criterion of response (Umberger, Gass and Curtis, 1958). An alternative method for estrogenizing poultry is the addition of dienestrol
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Greenblatt, R. B., and N. H. Brown, 19S2. The storage of estrogen in human fat after estrogen administration. Am. J. Gynec. Obstet. 61: 13611363. Hooker, C. W., 1945. A criterion of luteal activity in the mouse. Anatomical Record, 93: 333-347. Jones, O., and F. E. Deatherage, 1953. The diethylstilbestrol content of the meat from chickens treated with this estrogenic substance. Food Research, 18: 30-34. Koch, W., and G. Heim, 1952. Die Speicherung von Oestrogenen im Tierkorper. I. Hormonal kastrierte Hahnchen. Endokrinologie, 29: 288-293. Lee, H. M., E. B. Robbins and K. K. Chen, 1942. The potency of stilbestrol in the immature female rat. Endocrinology, 30: 469-473. Lorenz, F. W., 1954. Effects of estrogens on domestic fowl and applications in the poultry industry. Vitamins and Hormones, 12: 235-275. Merker, P. C , L. D. Edwards, F. N. Andrews and J. E. Christian, 1955. The estrogenic activity of residues obtained from chickens treated with pellets of diethylstilbestrol. Poultry Sci. 34:11181125. Meyer, R. K., 1952. Endocrine Laboratories Report No. 1EL-270. In U. S. Congress, House. Select Committee to Investigate the Use of Chemicals in Food and Cosmetics, 82nd Congress, 2nd Session. Hearings pursuant to H. Res. 74 and H. Res. 447. Part 3, Appendix B, pp. 1436-1438. Rubin, B. L., A. S. Dorfman, L. Black and R. I. Dorfman, 1951. Bioassay of estrogens using the
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