Assessment of human estrogen receptor agonistic/antagonistic effects of veterinary drugs used for livestock and farmed fish by OECD in vitro stably transfected transcriptional activation assays.

The presence of veterinary drug residues in foods and the environment could potentially cause adverse effects on humans and wildlife. Several veterinary drugs were reported to exhibit endocrine disrupting effects via binding affinities to sexual hormone receptors such as estrogen and androgen receptors. Therefore, we confirmed the human estrogen receptor (ER) agonistic/antagonistic effects of 135 chemicals that were used as veterinary drugs in Korea by the official Organization for Economic Cooperation and Development (OECD) in vitro ER transcriptional activation (TA) assay using the VM7Luc4E2 cell line. In the case of ER agonist screening, 7 veterinary drugs (cefuroxime, cymiazole, trenbolone, zeranol, phoxim, altrenogest and nandrolone) were determined to be ER agonists. In addition, only zeranol was found to exhibit weak ER antagonistic activity. These 7 veterinary drugs, which were determined as ER agonists and/or antagonists by an OECD in vitro assay, were also found to have binding affinity to ERs. These results indicate that various veterinary drugs possess potential (anti-)estrogenic effects. However, further study is needed to determine the precise endocrine-disrupting effects of these compounds.

Pharmacologic activation of estrogen receptor ß increases mitochondrial function, energy expenditure, and brown adipose tissue.

Most satiety-inducing obesity therapeutics, despite modest efficacy, have safety concerns that underscore the need for effective peripherally acting drugs. An attractive therapeutic approach for obesity is to optimize/maximize energy expenditure by increasing energy-utilizing thermogenic brown adipose tissue. We used in vivo and in vitro models to determine the role of estrogen receptor ß (ER-ß) and its ligands on adipose biology. RNA sequencing and metabolomics were used to determine the mechanism of action of ER-ß and its ligands. Estrogen receptor ß (ER-ß) and its selective ligand reprogrammed preadipocytes and precursor stem cells into brown adipose tissue and increased mitochondrial respiration. An ER-ß-selective ligand increased markers of tricarboxylic acid-dependent and -independent energy biogenesis and oxygen consumption in mice without a concomitant increase in physical activity or food consumption, all culminating in significantly reduced weight gain and adiposity. The antiobesity effects of ER-ß ligand were not observed in ER-ß-knockout mice. Serum metabolite profiles of adult lean and juvenile mice were comparable, while that of adult obese mice was distinct, indicating a possible impact of obesity on age-dependent metabolism. This phenotype was partially reversed by ER-ß-selective ligand. These data highlight a new role for ER-ß in adipose biology and its potential to be a safer alternative peripheral therapeutic target for obesity.-Ponnusamy, S., Tran, Q. T., Harvey, I., Smallwood, H. S., Thiyagarajan, T., Banerjee, S., Johnson, D. L., Dalton, J. T., Sullivan, R. D., Miller, D. D., Bridges, D., Narayanan, R. Pharmacologic activation of estrogen receptor ß increases mitochondrial function, energy expenditure, and brown adipose tissue.

Effects of postnatal estrogen manipulations on juvenile alloparental behavior.

Sex- and species-specific patterns of estrogen receptor (ER)-α expression are established early in development, which may contribute to sexual differentiation of behavior and determine male social organization. The current study investigated the effects of ERα and ERß activation during the second postnatal week on subsequent alloparental behavior and ERα expression in juvenile prairie voles. Male and female pups were treated daily with 17ß-estradiol (E2, ERα/ERß agonist), PPT (selective ERα agonist), DPN (selective ERß agonist), or the oil vehicle on postnatal days (PD) 8-14. Alloparental behavior and ERα expression were examined at PD21. PPT treatment inhibited prosocial motivation in males and increased pup-directed aggression in both sexes. E2 and DPN had no apparent effect on behavior in either sex. PPT-treated males had increased ERα expression in the medial preoptic area (MPN), medial amygdala (MEApd) and bed nucleus of the stria terminalis (BSTpr). DPN treatment also increased ERα expression in males, but only in the BSTpr. Female ERα expression was unaffected by treatment. These results support the hypothesis that ERα activation in early life is associated with less prosocial patterns of central ERα expression and alloparental behavior in males. The lack of an effect of E2 on behavior suggests that ERß may antagonize the effects of ERα on alloparental behavior. The results in DPN-treated males suggest that ERα in the MEApd, and not the BSTpr, may be a primary determinant of alloparental behavior in males.

Population pharmacokinetic/pharmacodynamic assessment of pharmacological effect of a selective estrogen receptor ß agonist on total testosterone in healthy men.

BACKGROUND: LY500307 is a highly selective estrogen receptor ß (ERß) agonist, which loses its selectivity at high dose and leads to undesirable suppression of total testosterone (TT) concentration. The objective of the present analysis was to define the LY500307 dose with minimal effect on TT METHODS: LY500307 and TT concentrations were obtained from a single ascending-dose study in a total of 30 healthy male subjects. LY500307 (in the range of 0.5 to 500 mg) or placebo was administered orally as a single dose on 2 occasions with a 3-week washout period. A population pharmacokinetics/pharmacodynamics (PK/PD) model that integrated Fourier series in an indirect response model was developed to describe the circadian rhythm of TT and the exposure-response relationship of LY500307 on TT. RESULTS: The maximum TT suppression (Emax ) was approximately 28.6%. The potency (EC50 ) of LY500307 on TT suppression was approximately 1.69 ng/mL with a 95%CI of 0.871 to 4.44 ng/mL. This model could provide inferences on LY500307 dose levels that would result in various magnitudes of TT suppression. CONCLUSIONS: Population PK/PD modeling is a highly sensitive tool to detect exposure-response relationships on top of the complicated and highly variable circadian rhythm of TT.

Fertility and developmental toxicity assessment in rats and rabbits with LY500307, a selective estrogen receptor beta (ERß) agonist.

LY500307 is a selective estrogen receptor beta (ERß) agonist that was developed for the treatment of benign prostatic hyperplasia. The in vitro functional selectivity of LY500307 for ERß agonist activity is 32-fold above the activity at the alpha receptor (ERα). LY500307 was evaluated in a series of male (M) and female (F) rat fertility and rat and rabbit embryo-fetal development (EFD) studies, using 20 or 25 animals/group. LY500307 was administered daily by oral gavage starting 2 weeks (F) or 10 weeks (M) before mating, during cohabitation, until necropsy (M) or through gestation day (GD) 6 (F) in the fertility studies and from GD 6 to 17 (rats) or GD 7 to 19 (rabbits) in the EFD studies. Dosage levels of LY500307 ranged from 0.03 to 10 mg/kg/day for rats and from 1 to 25 mg/kg/day for rabbits. Fertility, estrous, maternal reproductive endpoints, conceptus viability, sperm parameters, organ weights, and histopathology were evaluated in the fertility studies. Maternal reproductive endpoints and fetal viability, weight, and morphology were evaluated in the EFD studies. Toxicokinetics were assessed in satellite animals. At 10 mg/kg/day in the male fertility study, findings included decreased body weight (BW); food consumption (FC); fertility, mating, and conception indices; sperm concentration; and reproductive tissue weight (associated with atrophic histologic changes). In the female fertility study, effects included decreased BW and FC at ≥0.3 mg/kg/day and persistent diestrus, delayed mating, and reduced fertility/conception indices at 3 mg/kg/day. In the rat EFD study, findings included decreased maternal BW and FC and increased incidences of adverse clinical signs, abortion, maternal mortality/moribundity, postimplantation loss, and fetal skeletal variations at 3 mg/kg/day. Effects in the rabbit EFD study were limited to decreases in maternal BW and FC at 25 mg/kg/day. In general, systemic maternal exposure increased proportionally with dosage in rats, but less than proportionally in rabbits. In conclusion, the no-observed adverse effect levels following LY500307 administration were 1 mg/kg/day for male rat fertility, 0.3 mg/kg/day for female rat fertility and EFD, and 25 mg/kg/day for rabbit EFD. Adverse reproductive and developmental effects only occurred at or above parentally toxic dosage levels and were considered predominantly due to off-target ERα effects.

The assessment of estrogenic or anti-estrogenic activity of chemicals by the human stably transfected estrogen sensitive MELN cell line: results of test performance and transferability.

The need for development and validation of in vitro hormone receptor transactivation assays as important alternative tools to study interactions with sex hormone receptors is outlined by international organisations, as such assays should be included in the OECD conceptual framework for the testing and assessment of endocrine active chemicals. Therefore as part of the European Union (EU)-sponsored 6th framework project ReProTect, the validation study with MELN cells, MCF-7 cells (ER+, estrogen receptor positive) which were stably transfected with the estrogen responsive gene ERE-betaGlob-Luc-SVNeo was set up. Standard operating procedures including a prescreen assay for unknown chemicals, an ER-agonist assay and an ER-antagonist assay were developed at the Flemish Institute for Technological Research, Belgium, and successfully transferred to Bayer Schering Pharma AG, Germany. Test results were obtained for 16 chemicals, and it was demonstrated that the MELN assay is transferable, robust and reproducible which allowed to rank chemical compounds according to their strong to weak affinity for the estrogen-alpha receptor, or identify negative chemicals within the test range up to 10(-5)M. Besides the screening for agonism, we demonstrated the suitability of MELN cells to test for antagonistic activity, which is of added value compared to current validated assays. As the MELN assay successfully passed the first modules of the ECVAM validation procedure, it now should be considered for further steps including the definition of a prediction model and application domain to get it accepted as an alternative screening assay, contributing to the 3R's with a reduction of animal experiments.