No abstract

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.

Methyl tert-butyl ether (MTBE) is an oxygenated fuel additive used to decrease carbon monoxide emissions during combustion. MTBE is a nongenotoxic chemical that induces Leydig cell tumors (LCT) in male rats. The mechanism of MTBE-induced LCT is not known; however, LCT induced by other nongenotoxic chemicals have been associated with the disruption of the hypothalamus-pituitary-testicular (HPT) axis. The objective of this study was to determine whether MTBE functions as an endocrine-active compound by affecting levels of specific hormones involved in the maintenance of the HPT axis. Nine-week-old male Sprague-Dawley rats were administered MTBE by gavage at 0, 250, 500, 1000, or 1500 mg MTBE/kg/day for 15 or 28 consecutive days and sacrificed 1 h following the last dose. Relative testis weights were increased only in high-dose animals treated for 28 days, and no testicular lesions were observed at any dose level. Adrenal gland, liver, and kidney weights were also increased. Histologic changes included protein droplet nephropathy of the kidney and centrilobular hypertrophy of the liver. Interstitial fluid and serum testosterone levels as well as serum prolactin levels were decreased only in animals treated with 1500 mg MTBE/kg/day for 15 days. At 28 days, serum triiodothyronine (T3) was significantly decreased at 1000 and 1500 mg MTBE/kg/day compared to control animals, and a decrease in serum luteinizing hormone and dihydrotestosterone was observed at 1500 mg MTBE/kg/day. These results indicate that MTBE causes mild perturbations in T3 and prolactin; however, the changes in testosterone and LH levels did not fit the pattern caused by known Leydig cell tumorigens.

tert-Butyl alcohol (TBA) is widely used in the manufacturing of certain perfumes, cosmetics, drugs, paint removers, methyl tert-butyl ether (MTBE), and industrial solvents. In both rodents and humans, TBA is a major metabolite of MTBE, an oxygenated fuel additive. Chronic TBA exposure causes protein droplet nephropathy, alpha2u-globulin (alpha2u) accumulation, renal cell proliferation, and with chronic exposure, renal tumors in male, but not female, rats. These effects suggest an alpha2u-mediated mechanism for renal tumors. The objective of the present study was to determine whether TBA or its metabolites bind to alpha2u. Mature male and female F-344 rats were administered a single gavage dose of 500 mg/kg TBA, 500 mg/kg (14)C-TBA, or corn oil. TBA equivalents/gram or ml of tissue in the male rat kidney, liver, and blood were higher than the levels measured in female rat tissue 12 h after (14)C-TBA administration. Gel filtration and anion-exchange chromatography demonstrated that (14)C-TBA-derived radioactivity co-eluted with alpha2u from male kidney cytosol. Protein dialysis studies demonstrated that the interaction between (14)C-TBA-derived radioactivity and alpha2u was reversible. Incubations of the low-molecular-weight protein fraction (LMWPF) isolated from (14)C-TBA-treated male rat kidneys with d-limonene oxide (a chemical with a high affinity to alpha2u) demonstrated that (14)C-TBA-derived radioactivity was displaced. Gas chromatography-mass spectrometry analysis confirmed that TBA was present in this LMWPF fraction. These results demonstrate that TBA interacts with alpha2u, which explains the accumulation of alpha2u in the male rat kidney following TBA exposure.

Methyl tert-butyl ether (MTBE) is an oxygenated fuel additive used to decrease carbon monoxide emissions during gasoline combustion. In the current study, we investigated the hypothesis that the MTBE-induced decrease in serum testosterone levels in male rats may be due in part to the ability of MTBE to induce the metabolism of endogenous testosterone and hence enhance its clearance. Nine-week-old male Sprague-Dawley rats were gavaged with 250, 500, 1000, or 1500 mg MTBE/kg/day in corn oil or corn oil alone for 15 or 28 consecutive days. Increased relative liver weight (10-14%) and minimal-to-moderate centrilobular hypertrophy were observed in rats treated with 1000 and 1500 mg MTBE/kg/day (high doses) for 28 days. Total hepatic microsomal cytochrome P450 (CYP) was increased 1. 3-fold in the high-dose, 15-day-treated rats. An evaluation of specific CYP activities using selective markers demonstrated a 2. 0-fold increase in CYP2B1/2 in rats treated with 1000 mg MTBE/kg/day for 28 days, and with 1500 mg MTBE/kg/day for 15 and 28 days (6.5- and 2.9-fold, respectively). CYP1A1/2, CYP2A1, and CYP2E1 activities were increased 1.5-, 2.4-, and 2.3-fold, respectively, in high-dose, 15-day-treated rats. CYP2E1 was also increased in high-dose, 28-day-treated rats (2.0-fold). CYP3A1/2 was increased 2.1-fold and UDP-glucuronosyltransferase activity 1.7-fold in high-dose, 28-day-treated rats. MTBE also induced its own metabolism 2.1-fold in high-dose, 28-day-treated rats. Results indicate that MTBE induces selected enzymes involved in testosterone metabolism. The decrease in serum testosterone observed following MTBE administration may be the result of enhanced testosterone metabolism and subsequent clearance.