Journul of Srumid Biorh~nisrr,r. Vol. I I. pp. 919 to 922 Pergamon Press Ltd 1979. Printed in Great Britain
ASPECTS ON THE FORMATION AND DETECTION OF TISSUE LEVELS OF ANABOLIC STEROIDS IN DOMESTIC ANIMALS B. HOFFMANN TU-Miinchen/Weihenstephan, Institut Wr Physiologic, 805 Freising, and Institut fur Veterinlrmedizin, Bundesgesundheitsamt, 1000 Berlin 33, Germany SUMMARY Anabolic agents used in animal production can be classified according to their biological activity into compounds with oestrogenic, androgenic and gestagenic activity and according to the structure into endogeneous steroids, extraneous steroids and non-steroidal compounds. Metabolism of endogenous steroids is well established, a major principle being the formation of biologically less active metabolites, whilst the metabolism is not yet fully understood for non-endogenous steroids. Differences between compounds exist and it can be assumed that those with a high oral activity are only to a small extent biodegraded. Tissue residues of anabolic steroids can be expected in the rig/g to pg/g range. By using radioimmunoassay techniques, these low level residues and the naturally occurring tissue-concentration of endogenous steroids can be quantitated. Depending on the. animal and the tissue, endogenous steroids exhibit large variations. Oestrone was >2 rig/g in muscle from a pregnant cow and, like oestradiol-17/?, generally ~0.1 rig/g in all tissues in veal-calves, heifers and steers. Testosterone was highest in the bull (0.5 &g-11 rig/g), then the heifer (0.1 ngjg-O.6rig/g) and veal-calf (0.02nelB_o.3rig/g). Progesterone in fat was > 300 rig/g in pregnant cows and 17 and 6 rig/g in non-pregnant heifers and calves respectively. After implantation of 20 mg oestradiol-17/J combined with 200 mg testosterone or progesterone into veal-calves 70-77 days before slaughter the oestrogen-tissue concentrations were co.1 rig/g and the testosterone and progesterone concentrations were slightly elevated but still lower or in the same range as in heifers. Implantation of 14Omg trienbolone acetate (TBA) into veal-calves 70-77 days before slaughter yielded residues of free trienbolone between 0.13 and 0.5 rig/g.. It is concluded that endogenous hormones are natural constitutents of edible animal tissue and that the amount of residues present in treated or untreated animal will not measurably contribute to the levels already seen in the human. In the case of extraneous compounds residues should be graded according to normal toxicological criteria.
From their use in the human, anabolic steroids are generally assumed to be compounds with androgenic activity some of which, like 19-nor-testosterone, are also used in animals (for example the race horse) to increase their athletic capabilities. In this situation, the only concern in respect to the use of these compounds is the problem of doping and adequate methodology to control for this situation has been described [l, 21. However of more general concern is that anabolic steroids or rather compounds with sex hormone-like activities (oestrogenic, androgenic and gestagenic activity) are routinely used in food producing animals to improve weight gain and feed efficiency [3]. This paper is an attempt to review some of the important qualitative and quantitative aspects in relation to the residue problem arising from the treatment of food animals with these anabolic steroids. ANABOLIC STEROIDS USED DOMESTIC
IN
ANIMALS
The type of anabolic preparations, their composition and field of application are listed in Table 1. Beside endogenous and extraneous steroids there is 919
a rather big block of non-steroidal compounds with oestrogenic activity, consisting of the stilbene derivatives and zeranol, the latter being a derivative of a myco-oestrogen [4]. At present, anabolic agents are predominantly used in ruminants and now they are hardly used in poultry [S]. The efficacy of anabolic preparations in animal production seems to be mainly related to the oestrogenic part of the drug [6,7]. Most of these preparations are given by implantation and the usual dose for the oestrogen ranges between 20-40 mg and 140-300 mg for the non-oestrogen. The suggested time of implantation generally varies between 60 to 90 days before slaughter [S]. ASPECTS
OF METABOLISM
This has been reviewed in detail elsewhere [9, lo] and therefore only some more important aspects will be mentioned. After exogenous application, endogenous steroids are biodegraded and eliminated by well-established pathways. In cattle 17Soestradiol as well as testosterone are to a large extent transformed into their biologically almost non-active 17a-epimers [IO, 111. For 17a-oestradiol it has been demonstrated that it is partly converted to a glycoside [12]. Elimin-
920
8. HOFFMANN Table 1. Composition of various anabolic preparations (taken from [9]) Preparation
Main component
Oestrogenic
Non-oestrogenic
part
Type of animal production
Endogenous steroids
17@Qestradiol ~7~-~s~adiol
Progesterone Testosterone
Veal calf Veal calf
Extraneous steroids
Oestradiolbenzoate Oestradiolbenzoate O~trad~oi-monopaImitate 17~-O~tradiol
Progesterone Testosteronepropionate
Steer, heifer, lamb Steer, heifer, lamb Poultry Veal calf/steer Heifer/cow
Non-steroidal compounds
Diethylstilbestrol (DES) DES DES Hexoestrol Dienoestroldiacetate Zeranol
ation occurs predominantly via the fecal route after having been subjected to enterohepatic circulation. Differing from endogenous steroids, extraneous steroids may or may not be catabolized to biologically less active compounds. It has been recently demonstrated in cattle, that trienbolone seems to be largely metabolized to its biologically less active 17a-epimer [13], but for example no such isolation on the biodegradation of DES is available [8]. These compounds, like DES, are eliminated basically unchanged with the bile after simple conjugation to glucuronic acid and become partly available again through the enteroheparic circulation, which may in part explain their high oral activity 193. It has been demonstrated for oestrogens and trienbolone that some residues seem to occur covalently bound with proteins [14,iS]; however, so far investigated only DES seems to bind to DNA [163. METXODS
OF DETECTION
As was clearly demonstrated by tracer studies with radiolabelled anabolic steroids, the residue concentrations to be expected after an adequate withdrawal period are in the low parts per billion and parts per million-range [9,17,18]. From the few methods available at present which are sensitive enough to measure these low residue concentrations, only radioimmunoassay has been applied on a broader basis. Various appiications of radioimmunoassay developed so far are shown in Table 2 (for methodological details refer to the original papers). It is generally acknowledged that the procedures involved in measuring steroid hormone concentrations in tissues are more laborious than the methods applied for plasma. Most difficulties are associated with the determination of low-level oestrogens. However, apparently due to recent progress qu~titation in a range of 5-50 pg/g tissue is now possible [Zl, Forchielli 1978, personal communication]. In order to avoid the problems associated with
Trien~Ionea~tate TBA
(TBA)
Steer/heifer/lamb/veal Steer/heifer Pig Steer/heifer/lamb Poultry Steer/heifer/lamb/veal
Testosterone Methylt~tosterone
calf
calf
of tissue residue it has also been suggested that the elimination of anabolic agents could be monitored by measuring their concentrations in urine and faeces. Adequate meth~ology has been developed for the determination of oestrogens and trienic steroids, using thin-layer chromatography [24,25]. analysis
TISSUE RESIDUE
DATA
The only species investigated so far in some detail is cattle. As is shown in Table 3, tissue concentrations of hormones vary not only between an’imals, depending on sex and reproductive status, but also within each animal. In case of a high endogenous steroid production, like testosterone in the bull, or after giving excessive dosages, as was done in the case of triehboloneacetate, highest residue con~ntrations are measured in fat. Otherwise maximum hormone levels
can be determined in liver and kidney. The kidney (in particular) Seems to bind testosterone specifically [22]. The data obtained so far for endogenous anabolic steroids clearly indicate that under physiological conditions there are 100-lOOO-fold differences between animals. In case of treatments with these endogenous anabolics the residue levels seen in veal calves, steers and heifers are not elevated or only slightly elevated above controls and the lower part of the physiological distribution curve. This includes Table
2.
Application of RIA for measuring hormonal residues in edible animal tissues
Hormone O~tradio~-17~ Oestrone Testosterone Progesterone TBA/TBOH* DES
Tissue examined
Reference
m, m, m,
I, I, 1,
k. k. k,
f f f
[i9,20,21] C19,W
; m,
2 -,
k, -.
: -
~231 [LaschStza 1978 unpublished data]
IX1 PI
m = muscle; k = kidney; I = liver; f = fat. + trienbolone
921
Tissue levels of anabolic steroids in domestic animals Table 3. Concentrations
(pg/g) of various anabolic agents in their free form in tissues of treated and untreated cattle
Tissue examined Compound determined Testosterone
Progesterone
Oestradiol-17/3
Animal Bull Heifer Veal calf Veal calf (Treated)*
Steer Heifer Oestrone
TBOH (trienbolone)
Liver
Kidney
535 92 16
749 193 39
2783 595 256
10950 250 685
70
47
685
340
Pregnant cow Heifer Veal calf Veal calf (Treated)? Pregnant cow Veal calf (Untreated and treatedj)
Pregnant cow Veal calf (Treated and untreated:) Steers
Steer11 Veal calfj Veal calT Veal calr*
Fat
Muscle
37&860
ASPECTS ON THE FORMATION AND DETECTION OF TISSUE LEVELS OF ANABOLIC STEROIDS IN DOMESTIC ANIMALS B. HOFFMANN TU-Miinchen/Weihenstephan, Institut Wr Physiologic, 805 Freising, and Institut fur Veterinlrmedizin, Bundesgesundheitsamt, 1000 Berlin 33, Germany SUMMARY Anabolic agents used in animal production can be classified according to their biological activity into compounds with oestrogenic, androgenic and gestagenic activity and according to the structure into endogeneous steroids, extraneous steroids and non-steroidal compounds. Metabolism of endogenous steroids is well established, a major principle being the formation of biologically less active metabolites, whilst the metabolism is not yet fully understood for non-endogenous steroids. Differences between compounds exist and it can be assumed that those with a high oral activity are only to a small extent biodegraded. Tissue residues of anabolic steroids can be expected in the rig/g to pg/g range. By using radioimmunoassay techniques, these low level residues and the naturally occurring tissue-concentration of endogenous steroids can be quantitated. Depending on the. animal and the tissue, endogenous steroids exhibit large variations. Oestrone was >2 rig/g in muscle from a pregnant cow and, like oestradiol-17/?, generally ~0.1 rig/g in all tissues in veal-calves, heifers and steers. Testosterone was highest in the bull (0.5 &g-11 rig/g), then the heifer (0.1 ngjg-O.6rig/g) and veal-calf (0.02nelB_o.3rig/g). Progesterone in fat was > 300 rig/g in pregnant cows and 17 and 6 rig/g in non-pregnant heifers and calves respectively. After implantation of 20 mg oestradiol-17/J combined with 200 mg testosterone or progesterone into veal-calves 70-77 days before slaughter the oestrogen-tissue concentrations were co.1 rig/g and the testosterone and progesterone concentrations were slightly elevated but still lower or in the same range as in heifers. Implantation of 14Omg trienbolone acetate (TBA) into veal-calves 70-77 days before slaughter yielded residues of free trienbolone between 0.13 and 0.5 rig/g.. It is concluded that endogenous hormones are natural constitutents of edible animal tissue and that the amount of residues present in treated or untreated animal will not measurably contribute to the levels already seen in the human. In the case of extraneous compounds residues should be graded according to normal toxicological criteria.
From their use in the human, anabolic steroids are generally assumed to be compounds with androgenic activity some of which, like 19-nor-testosterone, are also used in animals (for example the race horse) to increase their athletic capabilities. In this situation, the only concern in respect to the use of these compounds is the problem of doping and adequate methodology to control for this situation has been described [l, 21. However of more general concern is that anabolic steroids or rather compounds with sex hormone-like activities (oestrogenic, androgenic and gestagenic activity) are routinely used in food producing animals to improve weight gain and feed efficiency [3]. This paper is an attempt to review some of the important qualitative and quantitative aspects in relation to the residue problem arising from the treatment of food animals with these anabolic steroids. ANABOLIC STEROIDS USED DOMESTIC
IN
ANIMALS
The type of anabolic preparations, their composition and field of application are listed in Table 1. Beside endogenous and extraneous steroids there is 919
a rather big block of non-steroidal compounds with oestrogenic activity, consisting of the stilbene derivatives and zeranol, the latter being a derivative of a myco-oestrogen [4]. At present, anabolic agents are predominantly used in ruminants and now they are hardly used in poultry [S]. The efficacy of anabolic preparations in animal production seems to be mainly related to the oestrogenic part of the drug [6,7]. Most of these preparations are given by implantation and the usual dose for the oestrogen ranges between 20-40 mg and 140-300 mg for the non-oestrogen. The suggested time of implantation generally varies between 60 to 90 days before slaughter [S]. ASPECTS
OF METABOLISM
This has been reviewed in detail elsewhere [9, lo] and therefore only some more important aspects will be mentioned. After exogenous application, endogenous steroids are biodegraded and eliminated by well-established pathways. In cattle 17Soestradiol as well as testosterone are to a large extent transformed into their biologically almost non-active 17a-epimers [IO, 111. For 17a-oestradiol it has been demonstrated that it is partly converted to a glycoside [12]. Elimin-
920
8. HOFFMANN Table 1. Composition of various anabolic preparations (taken from [9]) Preparation
Main component
Oestrogenic
Non-oestrogenic
part
Type of animal production
Endogenous steroids
17@Qestradiol ~7~-~s~adiol
Progesterone Testosterone
Veal calf Veal calf
Extraneous steroids
Oestradiolbenzoate Oestradiolbenzoate O~trad~oi-monopaImitate 17~-O~tradiol
Progesterone Testosteronepropionate
Steer, heifer, lamb Steer, heifer, lamb Poultry Veal calf/steer Heifer/cow
Non-steroidal compounds
Diethylstilbestrol (DES) DES DES Hexoestrol Dienoestroldiacetate Zeranol
ation occurs predominantly via the fecal route after having been subjected to enterohepatic circulation. Differing from endogenous steroids, extraneous steroids may or may not be catabolized to biologically less active compounds. It has been recently demonstrated in cattle, that trienbolone seems to be largely metabolized to its biologically less active 17a-epimer [13], but for example no such isolation on the biodegradation of DES is available [8]. These compounds, like DES, are eliminated basically unchanged with the bile after simple conjugation to glucuronic acid and become partly available again through the enteroheparic circulation, which may in part explain their high oral activity 193. It has been demonstrated for oestrogens and trienbolone that some residues seem to occur covalently bound with proteins [14,iS]; however, so far investigated only DES seems to bind to DNA [163. METXODS
OF DETECTION
As was clearly demonstrated by tracer studies with radiolabelled anabolic steroids, the residue concentrations to be expected after an adequate withdrawal period are in the low parts per billion and parts per million-range [9,17,18]. From the few methods available at present which are sensitive enough to measure these low residue concentrations, only radioimmunoassay has been applied on a broader basis. Various appiications of radioimmunoassay developed so far are shown in Table 2 (for methodological details refer to the original papers). It is generally acknowledged that the procedures involved in measuring steroid hormone concentrations in tissues are more laborious than the methods applied for plasma. Most difficulties are associated with the determination of low-level oestrogens. However, apparently due to recent progress qu~titation in a range of 5-50 pg/g tissue is now possible [Zl, Forchielli 1978, personal communication]. In order to avoid the problems associated with
Trien~Ionea~tate TBA
(TBA)
Steer/heifer/lamb/veal Steer/heifer Pig Steer/heifer/lamb Poultry Steer/heifer/lamb/veal
Testosterone Methylt~tosterone
calf
calf
of tissue residue it has also been suggested that the elimination of anabolic agents could be monitored by measuring their concentrations in urine and faeces. Adequate meth~ology has been developed for the determination of oestrogens and trienic steroids, using thin-layer chromatography [24,25]. analysis
TISSUE RESIDUE
DATA
The only species investigated so far in some detail is cattle. As is shown in Table 3, tissue concentrations of hormones vary not only between an’imals, depending on sex and reproductive status, but also within each animal. In case of a high endogenous steroid production, like testosterone in the bull, or after giving excessive dosages, as was done in the case of triehboloneacetate, highest residue con~ntrations are measured in fat. Otherwise maximum hormone levels
can be determined in liver and kidney. The kidney (in particular) Seems to bind testosterone specifically [22]. The data obtained so far for endogenous anabolic steroids clearly indicate that under physiological conditions there are 100-lOOO-fold differences between animals. In case of treatments with these endogenous anabolics the residue levels seen in veal calves, steers and heifers are not elevated or only slightly elevated above controls and the lower part of the physiological distribution curve. This includes Table
2.
Application of RIA for measuring hormonal residues in edible animal tissues
Hormone O~tradio~-17~ Oestrone Testosterone Progesterone TBA/TBOH* DES
Tissue examined
Reference
m, m, m,
I, I, 1,
k. k. k,
f f f
[i9,20,21] C19,W
; m,
2 -,
k, -.
: -
~231 [LaschStza 1978 unpublished data]
IX1 PI
m = muscle; k = kidney; I = liver; f = fat. + trienbolone
921
Tissue levels of anabolic steroids in domestic animals Table 3. Concentrations
(pg/g) of various anabolic agents in their free form in tissues of treated and untreated cattle
Tissue examined Compound determined Testosterone
Progesterone
Oestradiol-17/3
Animal Bull Heifer Veal calf Veal calf (Treated)*
Steer Heifer Oestrone
TBOH (trienbolone)
Liver
Kidney
535 92 16
749 193 39
2783 595 256
10950 250 685
70
47
685
340
Pregnant cow Heifer Veal calf Veal calf (Treated)? Pregnant cow Veal calf (Untreated and treatedj)
Pregnant cow Veal calf (Treated and untreated:) Steers
Steer11 Veal calfj Veal calT Veal calr*
Fat
Muscle
37&860
