Characterization of urinary metabolites of testosterone, methyltestosterone, mibolerone and boldebone in greyhound dogs

Androgenic steroids are used in female greyhound dogs to prevent the onset of estrus; moreover, these steroids also have potent anabolic activity. As anabolic steroids increase muscle mass and aggression in animals, the excessive use of these agents in racing greyhounds gives an unfair performance advantage to treated dogs. The biotransformation of most anabolic steroids has not been determined in greyhound dogs. The objective of the present study was to identify the urinary metabolites of testosterone, methyltestosterone, mibolerone, and boldenone in greyhound dogs. These steroids were administered orally (1 mg/kg) to either male or female greyhound dogs and urine samples were collected pre-administration and at 2, 4, 8, 12, 24, 72, and 96 h post-administration. Urine extracts were analyzed by high-performance liquid chromatography/mass spectrometry (HPLC/MS) to identify major metabolites and to determine their urinary excretion profiles. Major urinary metabolites, primarily glucuronide, conjugated and free, were detected for the selected steroids. Sulfate conjugation did not appear to be a major pathway for steroid metabolism and excretion in the greyhound dog. Phase I biotransformation was also evaluated using greyhound dog liver microsomes from untreated dogs. The identification of several in vivo steroid metabolites generated in this study will be useful in detecting these steroids in urine samples submitted for drug screening.     

Metabolism of endogenous and exogenous anabolic agents in cattle

The endogenous anabolic agents, estradiol-17 beta, progesterone and testosterone are steroids that are quickly metabolized by the liver and are not very active when administered orally. Estradiol-17 beta is excreted by the bovine in bile as free estradiol-17 alpha, and by swine, in urine, as glucuronides and sulfates. In ruminants, the primary metabolite of progesterone is pregnandiol and that of testosterone is epitestosterone. In horses, the metabolites of these compounds are primarily 17 ketosteroids. Esters of the endogenous anabolic agents are rapidly hydrolyzed and the nonesterified forms follow the same biotransformation pathways as the natural compounds biosynthesized by the animal. The exogenous anabolic agents, such as trenbolone acetate, zeranol and diethylstilbestrol, may be active by the oral route and are less readily metabolized in the liver than the endogenous anabolics. Their metabolic pathways may be complex and lead to excreted forms after glucuronide conjugation. With respect to biochemical mechanism of action, it can be assumed that the anabolics act like all steroids by way of intracellular receptors. Biotransformations could lead to more reactive molecules that may bind themselves to normal constituents of the organism. Bound metabolites are generally formed later than free metabolites, and are considered less toxic with a lower level of bioavailability.     

Metabolism of Methenolone Acetate in a Veal Calf

The use of anabolic steroids has been banned in the European Union since 1981. In this study, the metabolism of the anabolic steroid methenolone acetate, was investigated in a male veal calf. After daily oral administration of methenolone acetate, three main metabolites were detected in both urine and faeces samples. Among these metabolites, α-methenolone was apparently the main one, but 1-methyl-5α-androstan-3,17-diol and 3α-hydroxy-1-methyl-5α-androstan-17-one were also observed. The parent compound was still detectable in faeces. As a consequence, abuse of methenolone acetate as growth promoter can be monitored by analysing urine and faeces samples. A few days after the last treatment, however, no metabolites were observed. α-Methenolone was detectable in urine until 5 days after the last treatment, but in faeces no metabolites were detectable after 3 days.     

Embryo resorption following administration of steroidal compounds to rats in mid pregnancy.

In the course of experiments on the effects of anabolic steroids on the myocardium of rat conceptuses, we found that subcutaneous implantation of 10 mg of estradiol, Dianabol or testosterone to rats in mid pregnancy, resulted in embryo resorption. Placental tissue was identified only in estradiol-treated rats which also demonstrated a large amount of serosanguineous fluid that dilated the horns considerably. The yellow nodules of placental attachment sites were represented histologically by cellular and vascular proliferations between the inner and outer layers of the myometrium. The nodular aggregates of cells had variable features according to the steroid administered. Neither decidual cells nor metrial glands that are reported to be the constituents of placental attachment sites were seen in our material. We conclude that anabolic steroids are potent agents for embryo resorption, and that the cells in the nodules of placental attachment sites are likely to be derived from the myometrium.     

A perspective on anabolics

Extract

The word anabolic is an adjective and used as such it describes the property of a substance to promote in living cells the process of building complex substances from simple ones. Thus anabolic steroids are those which favour synthetic metabolism. The best known of these is testosterone, an androgen. Other anabolic steroids may be distinctly oestrogenic and some anabolic chemicals are not steroid hormones at all. Most anabolic substances are complex in theiractivity and, depending upon their primary nature, have the potential to produce undesirable physiological and/or behavioural side effects.     

Fertility of young mares after long-term anabolic steroid treatment

The effect of prior treatment with anabolic steroids was studied in 46 three-year-old mares. In the preceding year, these mares had been assigned to 1 of 4 treatment groups and had received the manufacturer's recommended dosage of 1.1 mg of boldenone undecylenate (BU)/kg of body weight, 4.4 mg of boldenone undecylenate (4 BU)/kg, 1.1 mg of nandrolone decanoate (ND)/kg, or 0.04 ml of sesame oil/kg (control, C). Mares had received an injection every 3 weeks for 54 weeks for a total of 19 injections, with the final injection in December. In the following breeding season, fewer (P less than 0.05) mares in all groups previously administered anabolic steroids displayed estrous behavior than did mares in the control group. Duration of estrus was shortened (P less than 0.05) in mares that had received steroids. Abnormal sexual behavior that was observed during steroid treatment continued (P less than 0.05) for up to 6 months after treatment ceased. However, observations of abnormal behavior declined with time (P less than 0.05). All mares in each treatment group ovulated by the end of the trial, and the interval to first ovulation was similar (P greater than 0.05). Ovarian size, follicular development, and conditions of the tubular genitalia was adversely (P less than 0.05) affected in mares in all steroid-treatment groups until approximately the middle of March. After that time, no difference was noted among groups. First-cycle pregnancy rates were 83%, 67%, 50%, and 42% for mares in the untreated, BU, 4 BU, and ND groups, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

An interlaboratory study of the pharmacokinetics of testosterone following intramuscular administration to Thoroughbred horses

Testosterone is an anabolic androgenic steroid (AAS) that is endogenously produced by both male and female horses that also has the potential for abuse when administered exogenously to race horses. To recommend appropriate withdrawal guidelines so that veterinarians can discontinue therapeutic use prior to competition, the pharmacokinetics and elimination of testosterone were investigated. An aqueous testosterone suspension was administered intramuscularly in the neck of Thoroughbred horses (n = 20). The disposition of testosterone from this formulation was characterized by an initial, rapid absorption phase followed by a much more variable secondary absorption phase. The median terminal half-life was 39 h. A second focus of this study was to compare the testosterone concentrations determined by two different laboratories using a percentage similarity model with a coefficient of variation of 16.5% showing good agreement between the two laboratories results. Based on the results of this study, a withdrawal period of 30 days for aqueous testosterone administered IM is recommended.     

Excretion mass balance evaluation,metabolite profile analysis and metabolite identification in plasma and excreta after oral administration of [14C]‐meloxicam to the male cat: preliminary study

Grudé, P., Guittard, J., Garcia, C., Daoulas, I., Thoulon, F., Ebner, T. Excretion mass balance evaluation, metabolite profile analysis and metabolite identification in plasma and excreta after oral administration of [¹⁴C]‐meloxicam to the male cat: preliminary study. J. vet. Pharmacol. Therap. doi: 10.1111/j.1365‐2885.2010.01157.x. The objective of this study was to investigate the metabolic pathways and routes of excretion of oral meloxicam in the cat. [¹⁴C]‐meloxicam was administered orally to three fasted male cats. Urine, faeces, vomit and cage washes were collected over the following 144 h period. Blood was collected predosing and at 3 and 12 h postdosing. Metabolites were identified by HPLC/MS/MS. When possible a metabolic structure was proposed for each metabolite detected. Only unchanged meloxicam was identified in plasma. Five major metabolites were detected in urine and four in faeces, which were identified by HPLC/MS/MS as products of oxidative metabolism. No conjugated metabolites were detected. Elimination occurred early (61% during the first 48 h). A total of 21% of the recovered dose was eliminated in urine (2% as unchanged meloxicam, 19% as metabolites) and 79% in the faeces (49% as unchanged meloxicam, 30% as metabolites). The results indicate that after oral administration the major route of excretion of meloxicam in the cat is faecal and that the main pathway of biotransformation of meloxicam in the cat is oxidation.     

Expression analysis of androgen-responsive genes in the prostate of veal calves treated with anabolic hormones

In order to identify indirect molecular biomarkers of anabolic treatments in veal calves, an animal experiment was performed using two combinations of growth promoters (consisting of boldenone undecylenate and estradiol benzoate, and of testosterone enantate and estradiol benzoate). We selected a set of 12 genes that are known to be androgen responsive in other mammalian species. The expression profile of this set of genes was analysed on prostate samples of veal calves using a real-time RT-PCR approach. For each selected gene the corresponding bovine sequence was obtained and a gene specific real-time assay was optimised and validated. The amplification was shown to be highly specific, linear and efficient. High reproducibility (<1%) and low-test variability (<2.5%) were also been achieved. Messenger RNA levels were quantified in prostate samples, non-parametric analysis of variance showed significant up-regulation of three genes (MAF, ESR1 and AR) and significant down-regulation of four genes (HMGCS1, HPGD, DBI, and LIM) in treated samples when compared with untreated controls. To assess the possibility of identifying hormone-treated animals by molecular means we performed a discriminant analysis that was effective in classifying treated and non-treated samples with an accuracy of 93%. Our results indicate that identification of treatment with steroid hormones in veal calves by means of gene expression analysis is a feasible approach and could be improved increasing both the number of genes and the number of controls analysed.     

The metabolism of labelled progesterone in the laying hen

Eight hours after administering 4–14C‐progesterone to laying hens four radioactive metabolites could be detected in the droppings. Excretion of labelled steroid products was still continuing 192 h after the injection. In blood a relatively high level of labelled progesterone was found 8 to 24 h after application. The last traces disappeared from blood after 72 h. Compared with mammals the hen metabolises progesterone at a slower rate.

Certain tissues were also examined 24 h after treatment. The adrenals showed a high level of labelled progesterone metabolites. The liver contained similar steroids as were found in the droppings. In the ovary, pituitary and skeletal muscle low or trace amounts of radioactive steroids were detected.     

Albendazole repeated administration induces cytochromes P4501A and accelerates albendazole deactivation in mouflon (Ovis musimon)

Adult mouflon ewes (Ovis musimon) were treated repeatedly with therapeutic doses of albendazole (ABZ, p.o. 7.5 mg/kg of body weight/day, for five consecutive days). Animals (treated or control) were sacrificed 24 h after the fifth dose of ABZ and liver and small intestine were collected to prepare microsomes. The activities of several biotransformation enzymes were measured in both hepatic and intestinal microsomes. A significant increase in the activity and amount of cytochromes P4501A (CYP1A) was observed in both tissues of ABZ treated mouflons compared to control animals. No other biotransformation enzymes tested were affected by five ABZ doses. The in vitro biotransformation of ABZ was studied in hepatic and intestinal microsomes from ABZ treated and control mouflons. Concentrations of two main ABZ metabolites - pharmacologically active ABZ sulfoxide and pharmacologically inactive ABZ sulfone were analysed using HPLC. A significant increase in rate of formation of ABZ sulfone (which is catalysed by CYP1A) was observed in hepatic as well as in intestinal microsomes from ABZ treated animals. The enhancement of ABZ deactivation by its repeated administration may affect the anthelmintic efficacy of this drug and may contribute to the development of parasite resistance.     

The effect of early postnatal treatment with a gonadotropin-releasing hormone agonist on the developmental profiles of testicular steroid hormones in the intact male pig

Three studies examined the effects of early postnatal treatment with a GnRH agonist on plasma concentrations of testosterone, dehydroepian-drosterone sulfate, 16-androstene steroids in fat and salivary glands, androstenone in fat and plasma, and testicular development of intact male pigs. The first study involved 45 7-d-old pigs assigned to three treatment groups: 1) boars administered 100 microg/kg of Lupron depot, 2) boars administered 200 microg/kg of Lupron depot, and 3) control boars receiving a saline carrier. The second study involved 20 7-d-old pigs assigned to two treatments: daily injection of 200 microL of 0.5 mg/mL Lupron from d 7 to 35 and controls treated with saline. The third study involved a total of 100 animals assigned to 10 groups of 10 based on their age at slaughter. These groups were subdivided into one of two treatments: 1) boars injected with 200 microL of 0.5 mg/mL of Lupron from d 3 to 35 and 2) control boars injected with saline. Testicular steroid hormone concentrations in plasma decreased (P < 0.01) within 7 d of GnRH agonist treatment. Following cessation of treatment, steroid levels increased to control levels and remained constant until the final rise at 5 mo. Plasma testosterone levels in the 100 microg/kg depot treatment group were higher (P < 0.05) than that of the 200 microg/kg and control group at 164 d of age. There were no differences between treatments (P > 0.05) in testicular steroid hormone levels at the end of study 2 or 3. There were no differences (P > 0.05) in concentrations of 16-androstene steroids in salivary glands between any of the treatment groups at market weight in studies 1 and 2. Fat androstenone levels measured in the third study ranged between 0.6 microg/g and 4.2 microg/g at 7 to 28 d of age. Treatment with GnRH agonist decreased plasma steroid levels and testicular development; however, by d 60 testicular size and weight were at control levels and remained similar until 180 d of age. The results of these studies indicate that daily administration of a GnRH agonist significantly decreased testicular development and steroidogenesis only during treatment, but testis growth and steroidogenesis had returned to control levels by 60 d of age in male pigs. Suppression of the early postnatal rise in testicular steroid hormones did not affect growth performance or steroid hormone levels at 5 to 6 mo of age.     

基于血浆非靶代谢组学探究急性笼养应激对蛋鸭代谢的影响

随着环保政策改革和养殖业的集约化、规模化发展,蛋鸭叠层式笼养模式的应用越来越广泛,笼养应激对蛋鸭的影响逐渐受到重视。本研究旨在利用非靶向代谢组学分析蛋鸭平养与笼养模式下血浆代谢物的差异。随机挑选平养组(floor-water rearing system, FWR组)和叠层式笼养组(cage-rearing system, CR组)蛋鸭各10只,跖骨静脉采血,通过高效液相色谱-质谱(HPLC-HRMS)非靶向代谢组学技术获得20个在不同饲养模式下的血浆代谢物图谱,经T检验等分析,取差异倍数(Fold-change)ratio≥2或ratio≤1/2、变量重要性(variable importance in projection, VIP)>1、q值<0.05为筛选条件得到显著差异代谢物并作通路富集分析。结果共筛选到差异代谢物30种,其中上调表达的差异代谢物14种(P<0.05),下调表达的差异代谢物16种(P<0.05)。差异代谢物种类主要有甘油磷脂(glycerophospholipids, 16种)、脂肪酰基(fatty acyls, 4种)、羧酸及其衍生物...     

Diagnosis, therapy and endocrinologic parameters of persistent follicles in mares in comparison with preovulatory follicles]

During the 1997 breeding season persistent follicles were diagnosed in 17 mares. In 16 of these mares a total of 17 follicles were transabdominally punctured and the steroids oestradiol, progesterone and testosterone were measured in the follicular fluid and in blood serum. In ten mares serving as a control group preovulatory follicles were punctured. The follicular fluid of the persistent follicles revealed a very high variability of the steroid concentrations. Depending on the steroid ratio within the follicles, eight follicles were rated as being intact, three follicles were undergoing atresia and five follicles were luteinized. Because of the high oestradiol levels of the follicular fluid within the control group, all of these follicles were considered to be intact. In both groups, no correlation of the steroid concentration between serum and follicular fluid was detectable. This fact argues against a passive diffusion of the steroids through the follicular wall. By puncturing the persistent follicles it was possible to bring the affected mares back into a physiological oestrus cycle within a normal dioestrus period.     

The absorption, distribution and excretion of anabolic agents

The metabolic fate of the anabolic agents, diethylstilbestrol, hexestrol, trenbolone acetate, zeranol and the endogenous steroids are discussed under the headings absorption, distribution and excretion. There is an optimum concentration of anabolic agent in the systemic circulation that results in a maximum increase in growth rate of farm animals. This optimum blood concentration should be maintained over a long period. However, there is rapid metabolism and excretion of anabolic agents with short half-lives in blood, and metabolic clearance rate equals entry rate. The rate of absorption of the agent, which is determined by formulation and site of administration, is most important and is best achieved by the use of slow release implants. The pattern of exponential absorption from compressed pellets of single anabolic agents is not ideal, and a more constant payout of drug, in particular estradiol-17 beta, is best achieved in combined preparations of agents or from silicone rubber implants impregnated with the agent. The high metabolic clearance rate and rapid excretion of anabolic substances influences the distribution of residues. Outside the site of administration, less than 1% of the administered dose is present in the animal. The lowest concentrations of residues are found in muscle and fat, higher concentrations are present in liver and kidney and the highest concentrations are in the bile, urine and feces.     

Effect of gonadal hormones on the plasma clearance and metabolite formation of antipyrine in the dwarf goat

The effect of gonadal hormones on the plasma elimination and urinary metabolite profile of antipyrine was studied in dwarf goats. Female goats were treated with testosterone and male goats were treated with 17β-oestradiol. Castrated males were treated with either testosterone or 17β-oestradiol. Antipyrine (25 mg/kg, i.v.) was given both before and after the hormonal treatments. The effects of the hormonal status on the plasma elimination of the parent compound were not consistent. This was possibly due to the fact that formation of the main metabolite of antipyrine in the goat, 4-hydroxy antipyrine (OHA), was not affected by sex or hormonal treatment. On the other hand, there were clear effects of hormonal status on urinary excretion of the three other metabolites. In females and castrated males testosterone suppressed the formation of norantipyrine (NORA), 3-hydroxymethylantipyrine (HMA) and 4,4'-dihydroxyantipyrine (DOHA). Intact males produced smaller amounts of these metabolites than females. It is concluded that distinct xenobiotic metabolizing pathways exist in the dwarf goat, which are influenced in their activity by gonadal hormones. This confirms previous findings in rats and mice. The possibility that sex hormones influence drug metabolism in food-producing animals could have consequences for veterinary therapeutics and public health. This study also demonstrates that, when using the antipyrine test for the assessment of hepatic drug metabolism, it is very important to include the determination of metabolites.     

The uptake of fenbendazole by cattle and buffalo following long-term low-level administration in urea-molasses blocks: Further studies on block formulations

Fenbendazole (Hoechst India Ltd.) was incorporated at 0.5 g/kg into urea molasses blocks made by two different processes. The proportion of the drug remaining in the blocks and the plasma concentrations of the parent compound and its metabolites were measured. Recovery of the drug in blocks made by the cold and the modified hot processes was 90% and 96%, respectively. The plasma metabolite profile revealed a plateau between days 4 and 6 of feeding in cattle and buffalo. However, the plasma concentrations of fenbendazole and its metabolites were low in buffalo compared to cattle.Abbreviations HPLC high-performance liquid chromatography - MUMB medicated urea molasses blocks - UMB urea-molasses block     

The metabolism of albendazole in the isolated perfused intestine of rats]

Pharmacokinetic studies on Albendazole after peroral administration demonstrate a rapid and complete biotransformation. The major metabolites Albendazole sulphoxide and -sulphone were detected in the plasma; the parent compound was only found sporadically at very low levels. These results indicate removal by the liver and/or the gut at first pass. After evidence is found that the liver has the capacity to sulphoxidize and to sulphonize Albendazole, biotransformation of the gut was examined using an isolated perfused rat gut model. A high-performance liquid chromatography method was used to simultaneously determinate Albendazole, -sulphoxide and -sulphone. Albendazole was biotransformed partly to Albendazole sulphoxide by the gut, whereby the metabolite but not the parent compound was absorbed. It is concluded, that the gut has the capacity to biotransform Albendazole. However, biotransformation is limited to the first step, the sulphoxidation.     

Plasma concentrations of testosterone and nandrolone in racing and nonracing intact male horses

Pennsylvania (PA) State Racing Commissions regulate the endogenous androgenic steroid, testosterone (TES), in racing intact males (RIM) by quantification of TES in post-race samples. Post-race plasma samples (2209) collected between March 2008 and November 2010 were analyzed for TES, nandrolone (NAN), and other anabolic steroids (ABS). Of the 2209 plasma samples, 2098 had quantifiable TES ≥ 25 pg/mL. Plasma (mean ± SD) concentrations of TES and NAN in RIM were 329.2 ± 266.4 and 96.0 ± 67.8 pg/mL, respectively. Only 64.6% of RIM had quantifiable concentration of NAN, and there was no relationship between TES and NAN. Plasma TES concentrations were significantly (P < 0.0001) higher during the months of April, May, June, July, and August. A significantly higher (P < 0.006) plasma TES was observed in Thoroughbred (TB) (347.6 ± 288.5 pg/mL) vs. that in Standardbred (STB) (315.4 ± 247.7 pg/mL). Plasma concentrations of TES from breeding stallions (BS) were 601.6 ± 356.5 pg/mL. Statistically significant (P < 0.0001) lower plasma concentrations of the two steroids were observed in RIM horses. Based on quantile distribution of TES in the RIM and BS populations, 99.5% were at or below 1546.1 and 1778.0 pg/mL, respectively. Based on this population of RIM, the suggested upper threshold plasma concentration of endogenous TES in horses competing in PA should remain at 2000 pg/mL.     

Effects of steroids on bovine T-lymphocyte blastogenesis in vitro

Day 13 to 24 bovine conceptuses convert C-19 and C-21 neutral steroids to 5 beta-reduced steroids with great efficiency. Pregnancy steroids have been reported to be immunomodulatory in several species. This study examined the possibility that 5 beta-reduced products of bovine conceptus steroid metabolism, precursors, related steroids, progesterone or cortisol might affect bovine T-cell blastogenesis. The steroids tested were 5 alpha-androstan-17 beta-ol-3-one, androstene-3, 17-dione, 5 beta-pregnane-3,20-dione and 5 beta-pregnane-3 alpha,20 alpha-diol. The ability of each steroid, over a dose range of 10(-2) to 10(4) nM, to affect phytohemagglutinin (PHA)-induced or concanavalin A (Con A)-induced bovine T cell blastogenesis, or the mixed lymphocyte reaction (MLR) was evaluated. Five lactating Holstein cows served as lymphocyte donors for mitogen studies and two, that exhibited strong MLR, were donors for the MLR evaluation. Of the seven steroids tested none affected blastogenesis in a dose-related manner except for cortisol, which suppressed Con A-induced lymphocyte transformation as well as the MLR. Cortisol did not affect PHA-induced blastogenesis. Thus, of the pregnancy steroids tested at the concentrations noted, none had significant immunomodulatory effects.