The gastrointestinal tract as target of steroid hormone action: quantification of steroid receptor mRNA expression (AR, ERalpha, ERbeta and PR) in 10 bovine gastrointestinal tract compartments by kinetic RT-PCR.

We have examined the tissue-specific mRNA expression pattern of androgen receptor (AR), both estrogen receptor (ER) subtypes ERalpha and ERbeta and progestin receptor (PR) in 10 bovine gastrointestinal compartments. Goal of this study was to evaluate the deviating tissue sensitivities and the influence of the estrogenic active preparation Ralgro on the compartment-specific expression regulation. Ralgro contains Zeranol which shows strong estrogenic and anabolic effects. Eight heifers were treated for 8 weeks with Ralgro at different dosages (0, 1, 3, and 10 times). To quantify the very low abundant steroid receptor mRNA transcripts sensitive and reliable real-time (kinetic) reverse transcription (RT)-PCR quantification methods were validated on the LightCycler. Expression results indicate the existence of AR and both ER subtypes in all 10 gastrointestinal compartments. PR receptor was expressed at very low abundancy. Gastrointestinal tissues exhibit a specific ERalpha and ERbeta expression pattern with high expression levels for both subtypes in rectum, colon and ileum. With increasing Zeranol concentrations a significant down-regulation for ERalpha and ERbeta was observed in jejunum (P<0.001 and <0.05, respectively). Significant up-regulations under estrogen treatment could be shown in abomasum for ERalpha (P<0.05) and in rectum for ERbeta (P<0.001). The authors conclude, that especially estrogens and the expression of their corresponding receptor subtypes may play an important role in the modulation and regulation in gastric as well as gut functions, cell proliferation and possibly in the pathophysiology of cell cancer. The different expression patterns of ERalpha and ERbeta can be regarded as support of the hypothesis that the subtype proteins may have different biological functions in the gastrointestinal tract. AR and PR seem to be not estrogen dependent.

Quantitative assessment of foetal exposure to trenbolone acetate, zeranol and melengestrol acetate, following maternal dosing in rabbits.

1. Residues of commonly used growth-promoting agents found in animal meat can be hormonally active and they have been implicated as possible endocrine disruptors in man. Although these compounds could be potentially detrimental to the developing foetus, it is not clear whether and to what extent they pass through placental barrier. 2. This issue was addressed using the rabbit as an animal model. Pregnant rabbits were treated with trenbolone acetate, zeranol or melengestrol acetate beginning at gestation day 14. Levels of active substances in plasma were screened by means of specific ELISA systems. The residues of parent compounds and their metabolites were quantified in maternal and foetal tissues on gestation day 27 using validated, sensitive HPLC/ELISA methods. 3. All three compounds crossed the placental barrier and were detectable in foetal tissues. The extent of tissue concentration varied depending on the compound and tissue analysed. Gender differences were observed in some instances.

Estradiol-17beta is produced in bovine corpus luteum.

The aim of this study was to investigate the expression of cytochrome P450 aromatase (aromatase) mRNA, its activity, and estradiol-17beta (estradiol) secretion in bovine corpus luteum (CL) during the estrous cycle. Expression of aromatase mRNA was examined in CL at the early, mid, late, and regressed luteal stages by using a reverse transcription-polymerase chain reaction. Aromatase mRNA was detected in all luteal stages examined, although aromatase expression was significantly lower during the early and regressed luteal phases compared to the mid and late luteal phases. Moreover, cultured midluteal cells clearly converted exogenous [(3)H]androstenedione into estradiol, and an aromatase inhibitor significantly inhibited this conversion. To characterize the local release of estradiol within the CL during the estrous cycle, an in vitro microdialysis system (MDS) of CL was conducted. Estradiol in MDS perfusate was confirmed by a reverse-phase high-performance liquid chromatography in combination with enzyme immunoassays. Basal release of estradiol from microdialyzed CL did not change during the estrous cycle. Additionally, when freshly prepared midluteal cells were exposed to estradiol (10(-14) to 10(-9) M), estradiol stimulated prostaglandin (PG) F(2alpha) secretion (P < 0.05), although it did not affect progesterone and oxytocin secretion. The overall results indicate that estradiol is produced locally in bovine CL throughout the estrous cycle, and they suggest that estradiol plays a role in regulating PGF(2alpha) production in CL as an autocrine/paracrine factor.

Studies on the antibody response of Lama glama--evaluation of the binding capacity of different IgG subtypes in ELISAs for clenbuterol and BSA.

Camelidae are known to produce three subtypes of immunoglobulin G (IgG), two of which are devoid of light chains. Two llamas (Lama glama) were immunised against clenbuterol-bovine serum albumin (BSA). Enzyme-linked immunosorbent assays (ELISAs) for clenbuterol and BSA on the basis of protein A-coated microtitration plates were established to investigate the titre development. Three subclasses of IgG (IgG(1): 29+66KDD, IgG(2): 52KDD, IgG(3): 56KDD) depending on their different binding properties to protein A and protein G could be separated chromatographically. Only IgG(1), which consists of conventional four-chain antibodies, bound to clenbuterol, whereas all forms of heavy-chain antibodies merely bound BSA.

Tissue-specific expression pattern of estrogen receptors (ER): quantification of ER alpha and ER beta mRNA with real-time RT-PCR.

We have examined the tissue-specific mRNA expression of ER alpha and ER beta in various bovine tissues using real-time RT-PCR. The goal of this study was to evaluate the deviating tissue sensitivities and the influence of the estrogenic active preparation RALGRO on the tissue-specific expression and regulation of both ER subtypes. RALGRO contains Zeranol (alpha-Zearalanol), a derivative of the mycotoxin Zearalenon, shows strong estrogenic and anabolic effects, and exhibits all symptoms of hyperestrogenism, in particular reproductive and developmental disorders. Eight heifers were treated over 8 weeks with multiple-dose implantations (0x, 1x, 3x, 10x) of Zeranol. Plasma Zeranol concentration, measured by enzyme immunoassay, of multiple treated heifers was elevated. To quantify ER alpha and ER beta transcripts also in low-abundant tissues, sensitive and reliable real-time RT-PCR quantification methods were developed and validated on the LightCycler. Expression results indicate the existence of both ER subtypes in all 15 investigated tissues. All tissues exhibited a specific ER alpha and ER beta expression pattern and regulation. With increasing Zeranol concentrations, a significant downregulation of ER alpha mRNA expression could be observed in jejunum (p<0.001) and kidney medulla (p<0.05). These data support the hypothesis that ER beta may have different biological functions than ER alpha, especially in kidney and jejunum.

Hormone contents in peripheral tissues after correct and off-label use of growth promoting hormones in cattle: effect of the implant preparations Filaplix-H, Raglo, Synovex-H and Synovex Plus.

Certain hormonal growth promoters are licensed in several beef producing countries outside the European Union (EU). Use in compliance with Good Veterinary Practice is mandatory. As risk assessment of hormone residues in animal tissues up to now has neglected potential off-label use, the present study dealt with two topics: 1) multiple treatment with the implant preparations Finaplix-H (200 mg trenbolone acetate), Ralgro (36 mg zeranol) and Synovex-H (200 mg testosterone propionate plus 20 mg estradiol benzoate) in heifers (1-fold, 3-fold and 10-fold dose), and 2) non-approved treatment of female veal calves (1-fold dose of Synovex-H or Synovex Plus with 200 mg trenbolone acetate plus 28 mg estradiol benzoate). Residues of estradiol-17beta, estradiol-17alpha, estrone and testosterone, trenbolone-17beta, trenbolone-17alpha and trendione or zeranol, respectively, were measured in loin, liver, kidney and peri-renal fat by high performance liquid chromatography/enzyme immunoassay (HPLC/EIA) after liquid-liquid extraction and solid-phase clean-up. The hormone residues in the multiple-dose experiments were dose-dependent and partially exceeded the threshold values: in the liver in one animal after 3-fold dose and in two animals after 10-fold dose of Finaplix-H, and in the liver and kidney after 3-fold and 10-fold dose of Synovex-H. Mean hormone residues in calves were mainly below those of heifers and did not infringe threshold values.

Detection of anabolic residues in misplaced implantation sites in cattle.

Eight weeks before slaughter, 26 heifers, 2 calves, and 1 steer were implanted with licensed anabolic preparations at off-label injection sites. After slaughter, 24 of 31 implantation sites (77%) were detected. Residual pellets of Revalor H contained a mean of 42.9 mg trenbolone acetate (range 19.8-57.7 mg) and 4.6 mg (1.96-6.45 mg) estradiol, corresponding to 30% (19.8-57.7%) and 32.7% (14.0-46.6%) of the originally applied dose, respectively. In the tissue areas containing residual Revalor H pellets, total residues ranged from 14.8 microg to 12.6 mg trenbolone acetate, 41.7 microg to 1.45 mg trenbolone, and 11.1 microg to 3.39 mg estradiol. The outer tissue areas of the injection sites contained <2 microg hormones. The preparations Synovex H, Finaplix H, Implus S, and Component EC behaved similarly to Revalor H. Residues of Synovex Plus were low, whereas the Compudose silicone rubber contained 58.8% of the implanted dose, but left no significant tissue residues. If implantation sites are processed in meat manufacturing, international threshold levels of the respective substances will be exceeded in tons of meat products.