Effects of early life exposure to flutamide on sexual development in Xenopus tropicalis

Sammanfattning: While endocrine disrupting chemicals have become a serious concern in human and environmental toxicology, many of the methods used for investigating developmental toxicity require long exposure periods, and thus can be technically challenging, labour intensive and expensive. For example, the current standard method, the Larval Amphibian Growth and Development Assay (LAGDA) has an exposure period of ³130 days. With the intent of developing a shorter assay for investigating amphibian developmental toxicity, the aim of this study was to characterise effects of the anti-androgenic model substance flutamide on reproductive development in the frog Xenopus tropicalis after a short 16-day post-metamorphosis exposure period using four dose groups: control (C), low (L), medium (M), and high (H) (0, 250, 500 and 1000 µg/L, nominal concentrations, n=58, 19, 39 and 36 respectively). Variations in body morphometry were used to compare C, L, M and H (for which there was no data available on sex ratios) and a random subsample of C (n=15) and H (n=15) individuals were analysed histologically for variation in germ cell maturity, gonadal cross sectional area, Müllerian Duct (MD) length and MD maturity. In addition to these endpoints, concentrations of the anti-Müllerian hormone (AMH), vitellogenin (VTG), oestradiol (E2) and testosterone (T) in the remainder of the body after dissection (normally discarded) for a subsample of C, M and H (N varying between 2 and 11 for AMH, VTG, E2 and T within the dose groups) were investigated with the objective of discovering whether these could be used as viable endpoints using commercially available ELISA kits. Due to random sampling there was an insufficient number of male H dose group individuals to allow for statistical analysis. In females, no significant differences in MD length, MD maturity and gonad size, were seen between the dose groups. No apparent differences in the proportion of mature germ cells in the testis were found, however females in the H dose group were found to have a significantly higher proportion of immature oocytes in the ovaries than C. The ELISA analysis was able to successfully detect AMH, VTG, E2 and T in all three dose groups. Some of the expected interrelationships between bodily AMH, VTG, E2 and T were observed in this study, whereas others were absent and others in direct opposition to prior research, suggesting this method shows promise but requires further study. While no conclusions cannot be drawn with regard to males from this study, effects attributable to flutamide can be detected in females during this dose period, and body homogenate ELISA shows promise for being a viable method but requires further research.