Developing predictive approaches to characterize adaptive responses of the reproductive endocrine axis to aromatase inhibition: I. Data generation in a small fish model.

Adaptive or compensatory responses to chemical exposure can significantly influence in vivo concentration-duration-response relationships. This study provided data to support development of a computational dynamic model of the hypothalamic-pituitary-gonadal axis of a model vertebrate and its response to aromatase inhibitors as a class of endocrine active chemicals. Fathead minnows (Pimephales promelas) were either exposed to the aromatase inhibitor fadrozole (0.5 or 30 µg/l) continuously for 1, 8, 12, 16, 20, 24, or 28 days or exposed for 8 days and then held in control water (no fadrozole) for an additional 4, 8, 12, 16, or 20 days. The time course of effects on ovarian steroid production, circulating 17ß-estradiol (E2) and vitellogenin (VTG) concentrations, and expression of steroidogenesis-related genes in the ovary was measured. Exposure to 30 µg fadrozole/l significantly reduced plasma E2 and VTG concentrations after just 1 day and those effects persisted throughout 28 days of exposure. In contrast, ex vivo E2 production was similar to that of controls on day 8-28 of exposure, whereas transcripts coding for aromatase and follicle-stimulating hormone receptor were elevated, suggesting a compensatory response. Following cessation of fadrozole exposure, ex vivo E2 and plasma E2 concentrations exceeded and then recovered to control levels, but plasma VTG concentrations did not, even after 20 days of depuration. Collectively these data provide several new insights into the nature and time course of adaptive responses to an aromatase inhibitor that support development of a computational model (see companion article).

Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor.

BACKGROUND: Several chemicals in the environment have the potential to inhibit aromatase, an enzyme critical to estrogen synthesis. OBJECTIVES: The objective of this study was to provide a detailed characterization of molecular and biochemical responses of female fathead minnows to a model aromatase inhibitor, fadrozole (FAD). METHODS: Fish were exposed via water to 0, 3, or 30 microg FAD/L for 8 days and then held in clean water for 8 days, with samples collected at four time points during each 8-day period. We quantified ex vivo steroid production, plasma steroids, and plasma vitellogenin (Vtg) concentrations and analyzed relative transcript abundance of 10 key regulatory genes in ovaries and 3 in pituitary tissue by real-time polymerase chain reaction. RESULTS: Ex vivo 17beta-estradiol (E2) production and plasma E2 and Vtg concentrations were significantly reduced after a single day of exposure to 3 microg or 30 microg FAD/L. However, plasma E2 concentrations recovered by the eighth day of exposure in the 3-microg/L group and within 1 day of cessation of exposure in the 30-microg/L group, indicating concentration- and time-dependent physiologic compensation and recovery. Concentration-dependent increases in transcripts coding for aromatase (A isoform), cytochrome P450 side-chain cleavage, steroidogenic acute regulatory protein, and follicle-stimulating hormone receptor all coincided with increased E2 production and recovery of plasma E2 concentrations. CONCLUSIONS: Results of this research highlight the need to consider compensation/adaptation and recovery when developing and interpreting short-term bioassays or biomarkers or when trying to predict the effects of chemical exposures based on mode of action.

Sex reversal of the amphibian, Xenopus tropicalis, following larval exposure to an aromatase inhibitor.

Aromatase is a steroidogenic enzyme that catalyzes the conversion of androgens to estrogens in vertebrates. Modulation of this enzyme's activity by xenobiotic exposure has been shown to adversely affect gonad differentiation in a number of diverse species. We hypothesized that exposure to the aromatase inhibitor, fadrozole, during the larval development of the tropical clawed frog, Xenopus tropicalis, would result in masculinization of the developing female gonad. Tadpoles were exposed to fadrozole at nominal concentrations from 1 to 64 microg/L in a flow-through system from < 24 h post-fertilization (Nieuwkoop Faber (NF) stage 15-20) to metamorphosis (NF stage 66). At metamorphosis, morphologically examined gonads indicated complete masculinization of all tadpoles at concentrations of 16 microg/L and above and a significant bias in sex ratio towards males at concentrations of 1 microg/L and above. No effects on time to metamorphosis, body mass, or body length were observed. A random subsample of frogs was raised to reproductive maturity (39 weeks post-fertilization) in control water. All frogs exposed as tadpoles to 16 microg/L fadrozole or greater possessed testes at sexual maturity. Intersexed gonads characterized by the presence of both testicular and ovarian tissue were observed in 12% of frogs in the 4 microg/L treatment. No differences in estradiol, testosterone, or vitellogenin plasma concentrations were observed in exposed males or females compared to controls. Females in the 4 microg/L treatment possessed a significantly greater percentage of pre-vitellogenic oocytes than controls and were significantly smaller in body mass. No differences in sperm counts were observed in exposed males compared to controls. Results from this study demonstrate that larval exposure to an aromatase inhibitor can result in the complete masculinization of female gonads. These masculinized females are phenotypically indistinguishable from normal males at adulthood. Lower levels of aromatase inhibition resulted in intersexed gonads and possible female reproductive impairment at adulthood. These results indicate that exposure of amphibians to xenobiotics capable of inhibiting aromatase would result in adverse reproductive consequences.

Successful detection of (anti-)androgenic and aromatase inhibitors in pre-spawning adult fathead minnows (Pimephales promelas) using easily measured endpoints of sexual development.

Screening assays have been successfully developed for the detection of (anti-)oestrogenic substances in several fish species, including the fathead minnow (Pimephales promelas). Previous work suggested that pre-spawning adult fathead minnows might be an appropriate life-stage for developing a screen to detect endocrine active substances (EASs). Pre-spawning adult fathead minnows, in which their phenotypic sex could be determined, were exposed in flow-through systems to three reference substances for 21 days, at 25 degrees C. Male and female fish, held in separate tanks, were exposed to dihydrotestosterone (DHT, androgen), flutamide (anti-androgen) and fadrozole (aromatase inhibitor). Nominal (mean measured) concentrations for DHT were 10 (6.0), 32 (6.1) and 100 (8.6) microg l(-1), for flutamide, 100 (95.3), 320 (320.4) and 1000 (938.6) microg l(-1) and for fadrozole, 25 (24.8), 50 (51.7) and 100 (95.5) microg l(-1). After 14 and 21 days exposure, fish were evaluated for growth, secondary sexual characteristics (SSCs, number and prominence of nuptial tubercles), gonadosomatic index (GSI) and plasma vitellogenin (VTG) concentrations. Development of nuptial tubercles was sensitive to both DHT and flutamide exposure. Exposure to DHT significantly increased the number of nuptial tubercles (male characteristic) in both males (more abundant) and females, after 14 days. Flutamide (938.6 microg l(-1), day 21) significantly reduced nuptial tubercle number in male fish. Fadrozole significantly inhibited ovarian growth (lower GSI) and significantly induced testis growth (51.7 and 95.5 microg l(-1)), after 21 days. Plasma VTG concentrations were significantly elevated in male fish (6.1 and 8.6 microg l(-1)), but inhibited in female fish (6.0 microg l(-1)), exposed to DHT. Flutamide had no effect on plasma VTG in male fish, but significantly induced VTG in female fish, after 21 days. Fadrozole significantly inhibited VTG in females and induced VTG synthesis in males, at day 21. These results show that SSCs, GSI and plasma VTG concentrations can be used in pre-spawning adult fathead minnows to screen for a range of classes of EASs. This work complements other published studies in supporting the current OECD effort towards validating a 21 days non-spawning fish screening assay for assessing (anti-)oestrogens, aromatase inhibitors and (anti-)androgens.