No abstract

The steroids trenbolone acetate (TbA) and melengestrol acetate (MGA) are licensed as growth promoters for farm animals in several meat-exporting countries. Although many studies have explored their safety for both animals and consumers, little is known about their fate after excretion by the animal. Our study aimed to determine the residues and degradation of trenbolone and MGA in solid dung, liquid manure, and soil. In animal experiments lasting 8 weeks, cattle were treated with TbA and MGA. Solid dung and, in case of trenbolone, liquid manure were collected and spread on maize fields after 4.5 and 5.5 months of storage, respectively. Determination of the hormone residues in all samples included extraction, clean-up (solid-phase extraction), separation of metabolites and interfering substances by HPLC (RP-18), and quantification by sensitive enzyme immunoassay. Procedures were validated by mass spectrometry (MS) methods. During storage of liquid manure the level of trenbolone decreased from 1,700 to 1,100 pg/g (17α-isomer), corresponding to a half-life of 267 days. Before storage, the concentrations in the dung hill ranged from 5 to 75 ng/g TbOH and from 0.3 to 8 ng/g MGA. After storage, levels up to 10 ng/g trenbolone, and 6 ng/g MGA were detected. In the soil samples trenbolone was traceable up to 8 weeks after fertilization, and MGA was detected even until the end of the cultivation period. The results show that these substances should be investigated further concerning their potential endocrine-disrupting activity in agricultural ecosystems.

Pathogens invading the mammary gland cause a complex signaling network that activates the early immune defense and leads to an outcome of inflammation symptoms. To examine the importance of mammary epithelial cells in these regulations and interactions resulting in a pathogen-related course of mastitis, we characterized the mRNA expression profile of key molecules of the innate immune system by quantitative real-time PCR. Mammary gland epithelial cells isolated on d 42 of lactation from 28 first-lactation Holstein dairy cows were cultured separately under standardized conditions and treated for 1, 6, and 24 h with heat-inactivated gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. Both pathogens increased mRNA expression patterns of proteins involved in pathogen recognition such as Toll-like receptors and nuclear factor-kappa B, whereas gram-negatives acted as a stronger stimulus. Furthermore, this could be confirmed by the expression profile of the proinflammatory cytokines tumor necrosis factor alpha, IL-1 beta, IL-6, and chemokines such as IL-8 and RANTES (regulated upon activation, normal T-cell expressed and secreted). Remarkably, at a low level of mRNA expression after 1 h of treatment these cytokines and chemokines were expressed at a significantly higher level in Staphyloccocus aureus than in Escherichia coli affected cells. Lactoferrin showed a deviating expression pattern to pathogen stimulation (i.e., at the 1-h measuring point Escherichia coli induced a higher mRNA expression, whereas the highest level was reached after 24 h of stimulation with Staphylococcus aureus). Complement factor 3 was the only measured factor that responded equally to both microorganisms. Our data emphasize the role of mammary epithelial cells in the immune defense of the udder and confirm their contribution to pathogen-related different courses of mastitis.

The steroids trenbolone acetate (TbA) and melengestrol acetate (MGA) are licensed as growth promoters for farm animals in several meat-exporting countries. Although many studies have explored their safety for both animals and consumers, little is known about their fate after excretion by the animal. Our study aimed to determine the residues and degradation of trenbolone and MGA in solid dung, liquid manure, and soil. In animal experiments lasting 8 weeks, cattle were treated with TbA and MGA. Solid dung and, in case of trenbolone, liquid manure were collected and spread on maize fields after 4.5 and 5.5 months of storage, respectively. Determination of the hormone residues in all samples included extraction, clean-up (solid-phase extraction), separation of metabolites and interfering substances by HPLC (RP-18), and quantification by sensitive enzyme immunoassay. Procedures were validated by mass spectrometry (MS) methods. During storage of liquid manure the level of trenbolone decreased from 1,700 to 1,100 pg/g (17alpha-isomer), corresponding to a half-life of 267 days. Before storage, the concentrations in the dung hill ranged from 5 to 75 ng/g TbOH and from 0.3 to 8 ng/g MGA. After storage, levels up to 10 ng/g trenbolone, and 6 ng/g MGA were detected. In the soil samples trenbolone was traceable up to 8 weeks after fertilization, and MGA was detected even until the end of the cultivation period. The results show that these substances should be investigated further concerning their potential endocrine-disrupting activity in agricultural ecosystems.

Pfaffl MW, Lange IG, Daxenberger A, Meyer HHD. Tissue-specific expression pattern of estrogen receptors (ER): Quantification of ERa and ERb mRNA with real-time RT-PCR. APMIS 2001;109: 345-55. We have examined the tissue-specific mRNA expression of ERa and ERb 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 (a-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 (0¿, 1¿, 3¿, 10¿) of Zeranol. Plasma Zeranol concentration, measured by enzyme immunoassay, of multiple treated heifers was elevated. To quantify ERa and ERb 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 ERa and ERb expression pattern and regulation. With increasing Zeranol concentrations, a significant downregulation of ERa mRNA expression could be observed in jejunum (pϽ0.001) and kidney medulla (pϽ0.05). These data support the hypothesis that ERb may have different biological functions than ERa, especially in kidney and jejunum.Cloning and sequencing of ERb in human (1), rat (2), and mouse (3) has provided the first example of a steroid hormone receptor existing in two isoforms, each of which is encoded by a separate gene. The ERb protein is smaller than the previously identified ERa (4, 5), but possesses the modular structure of distinct functional domains (A-F) characteristic of members of the nuclear receptors. The DNA-binding domain of ERa