Intramolecular [2 + 2] Photocycloaddition of Altrenogest: Confirmation of Product Structure, Theoretical Mechanistic Insight, and Bioactivity Assessment.

While studying the environmental fate of potent endocrine-active steroid hormones, we observed the formation of an intramolecular [2 + 2] photocycloaddition product (2) with a novel hexacyclic ring system following the photolysis of altrenogest (1). The structure and absolute configuration were established by X-ray diffraction analysis. Theoretical computations identified a barrierless two-step cyclization mechanism for the formation of 2 upon photoexcitation. 2 exhibited progesterone, estrogen, androgen, and pregnane X receptor activity, albeit generally with reduced potency relative to 1.

Use of gene expression, biochemical and metabolite profiles to enhance exposure and effects assessment of the model androgen 17ß-trenbolone in fish.

The impact of exposure by water to a model androgen, 17ß-trenbolone (TRB), was assessed in fathead minnows using an integrated molecular approach. This included classical measures of endocrine exposure such as impacts on testosterone (T), 17ß-estradiol (E2), and vitellogenin (VTG) concentrations in plasma, as well as determination of effects on the hepatic metabolome using proton nuclear magnetic resonance spectroscopy. In addition, the rates of production of T and E2 in ovary explants were measured, as were changes in a number of ovarian gene transcripts hypothesized to be relevant to androgen exposure. A temporally intensive 16-d test design was used to assess responses both during and after the TRB exposure (i.e., depuration/recovery). This strategy revealed time-dependent responses in females (little impact was seen in the males), in which changes in T and E2 production in the ovary, as well as levels in plasma, declined rapidly (within 1 d), followed shortly by a return to control levels. Gene expression measurements revealed dynamic control of transcript levels in the ovary and suggested potential mechanisms for compensation during the exposure phase of the test. Proton nuclear magnetic resonance spectroscopy revealed a number of hepatic metabolite changes that exhibited strong time and dose dependence. Furthermore, TRB appeared to induce the hepatic metabolome of females to become more like that of males at both high test concentrations of TRB (472 ng/L) and more environmentally relevant levels (33 ng/L).

Growth-promotant implants: managing the tools.

Great contemplation, conversation, and controversy have surrounded the use of growth-promotant implants since their inception in the 1950s. Since the very beginning, the purpose of growth promotants has been to enhance production efficiency, reduce the cost of production, and improve profitability. Changes in our understanding of the physiologic mechanisms involved in growth promotion have not altered this fundamental purpose. With enhanced knowledge of the impact of various compounds and doses on different classes of animals, and with the introduction of numerous products providing those compounds and doses, planning implant programs has become difficult. However, the net return from a well-designed implant program may mean the difference between profit and loss on a given set of cattle.

Comparison of alternative beef production systems based on forage finishing or grain-forage diets with or without growth promotants: 1. Feedlot performance, carcass quality, and production costs.

Forty Angus-cross steers were used to evaluate 5 beef cattle management regimens for their effect on growth performance, carcass characteristics, and cost of production. A 98-d growing phase was incorporated using grass silage with or without growth promotants (trenbolone acetate + estradiol implants, and monensin in the feed) or soybean meal. Dietary treatments in the finishing phase were developed, with or without addition of the same growth promotants, based on exclusive feeding of forages with minimal supplementation or the feeding of barley-based diets. Overall, ADG for animals treated with growth promotants or fed supplemented diets (soybean meal and barley) was increased (P < 0.01) by 25 and 21%, respectively, compared with steers reared on grass silage alone and not treated with growth promotants. Except for HCW (P < 0.01), the use of growth promotants did not affect carcass measurements. Increasing the proportion of barley in the diet of steers finished on forage produced a heavier HCW (P < 0.01) and a greater (P < 0.01) quality grade. Because of their lower HCW and quality grade, cattle targeted to a forage-fed, nonimplanted beef market would need to garner a 16% premium to be economically competitive with cattle finished conventionally.

Growth performance and shedding of some pathogenic bacteria in feedlot cattle treated with different growth-promoting agents.

Eighty steers with a mean body weight of 319 kg were used in a study to evaluate the effect of a growth-promoting implant (trenbolone acetate plus estradiol benzoate), monensin, and oxytetracycline on the steer performance and shedding of some foodborne pathogens. The steers were allotted to one of eight treatment combinations according to a randomized complete block design with 16 pens of five animals. Rectal fecal samples were collected before treatment commenced and over a period of more than 24 weeks to study the influence of treatments on the intestinal microbiology of the animals. Results supported the beneficial effect of the hormonal implant on the performance of feedlot steers (average daily gain, feed efficiency, and fat thickness), on carcass characteristics (hot carcass weight, lean yield), and economic value of the carcasses (P < 0.01). The levels of Escherichia coli in feces were not affected by treatments but remained high throughout the study period. Antibiotic-resistant isolates of E. coli were more frequently found as the study progressed but were not associated with any specific treatment. Also independently of treatment, we observed a reduction over time in the shedding of Campylobacter and Yersinia during the feeding period, whereas the shedding of Enterococcus was increased. The results of this study confirmed the beneficial economic effect of growth-promoting agents in beef production and showed that the agents tested did not specifically affect the overall microbial evolution of the animal gut. However, the study also showed, independently of the growth promoter used, the shedding of Campylobacter, Yersinia, and antibiotic-resistant E. coli in the feedlot environment. These bacteria also may be found in the colonic tissue of steers at slaughter and might be a source of carcasses contamination.

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.

Quantitative and qualitative assessment of milk production after pharmaceutical induction of lactation in the mare.

The induction of lactation is performed in ruminants by steroidogenic impregnation, followed by drugs intended to increase prolactin secretion. The aim of this study was to induce lactation in barren mares and to evaluate milk production. Five treated and 5 control mares were used in June and September in year 1, and 12 mares were used in year 2. Mares were administered a vaginal pessary (500 mg altrenogest and 50 mg estradiol benzoate) for 1 week. The 2nd week, another sponge with 100 mg estradiol benzoate was administered, together with 50 mg/100 kg body weight (BW) sulpiride in oil (IM q12h). All mares were milked by hand. Drug treatment was stopped after I L was obtained. Milk production and composition and plasma prolactin concentration were measured. In year 2, the same steroid treatment was applied, but mares received sulpiride (n = 6) or domperidone (1.1 mg/kg PO q12h) (n = 6). A milking machine and oxytocin injections 1 minute before the start of milking were used. In year 1, all treated mares started milking within 1-5 days after sulpiride treatment. Mean daily milk production was 0.88 +/- 0.52 L/500 kg BW. Milk immunoglobulin G (IgG) contents increased in all mares (IgG concentration range, 14-92 g/L). Plasma prolactin increased during sulpiride treatment (range. 27.7 +/- 2.9 to 43.7 +/- 6.7 ng/mL [before] to 289.0 +/- 7.8 ng/mL during treatment, P < .001). In year 2, results were similar to those in year 1, with peak IgG concentrations ranging from 4.2 to 106.7 g/L and a larger daily milk production (3.13 +/- 0.75 with sulpiride and 3.45 +/- 0.51 L/500 kg BW with domperidone). In conclusion, lactation can be induced in mares within 2 weeks, and some mares produce good-quality colostrum.

Assessment of oestrogenic potency of chemicals used as growth promoter by in-vitro methods.

Three in-vitro bioassays were used to compare the oestrogenic potency of chemicals used as growth promoter in beef cattle in certain non-European Union countries (17beta-oestradiol, alpha-zearalanol, testosterone, trenbolone, trenbolone acetate, melengestrol acetate) or found as food contaminant such as the mycotoxin zearalenone and some of their metabolites (17alpha-oestradiol, oestrone, 17alpha-epitestosterone, 19-nortestosterone, androstendione, zearalanone, alpha-zearalanol, beta-zearalanol, alpha-zearalenol, beta-zearalenol). The strong oestrogens 17alpha-ethinyl oestradiol and diethylstilboestrol were used as standards. The first bioassay was based on the activation of a reporter gene by oestrogens in recombinant yeast expressing human or rainbow trout oestrogen receptor. In the second bioassay, the vitellogenin gene induction of rainbow trout hepatocyte cultures was used as a biomarker for the exposure to oestrogens. The third bioassay was based on the alkaline phosphatase gene induction by oestrogens in the human endometrial Ishikawa cell line. The assessment of oestrogenic potency of these chemicals clearly demonstrates the strong oestrogenicity of the mycotoxin zearalenone and its metabolites and particularly alpha-zearalenol which was as potent as ethinyl oestradiol and diethylstilboestrol in the human endometrial Ishikawa cell line.

Assessment of estradiol and its metabolites in meat.

Most studies related to research on steroids in main edible tissues (muscle, liver or kidney) have focused on measurement of parent or major metabolite residues. In order to evaluate the estradiol content in bovine edible tissues, a multi-step extraction procedure was developed in conjunction with parallel metabolism studies of [14C]-17beta-estradiol in cattle (1-2). Various classes of free estradiol and conjugates were separated: estradiol -17beta and -17alpha, estradiol-17-fatty acid esters, estradiol 17-glycoside, estradiol 3-glucuronide, estradiol-17-glycoside and 3- glucuronide (diconjugates) were separated. No sulphates conjugated forms have been found at the detection level of the method. The quantification was realized by calibration with deuterated 17beta -estradiol -d3 standard and was validated at the ng x kg(-1) (ppt) level. Muscle, liver, kidney and fat samples from control or Revalor S single (licensed implantation) or multi-implanted steers have been assayed. The results show a wide variation between animals, but both the highest value and the mean of total estradiol content in each group proportionally increase from untreated to multi-implanted animals. In accordance with international rules, a calculation of the daily food supply of estradiol by such edible tissues in comparison with the acceptable daily intake was performed.