A time-course analysis of effects of the steroidogenesis inhibitor ketoconazole on components of the hypothalamic-pituitary-gonadal axis of fathead minnows

The objective of this study was to evaluate temporal effects of the model steroidogenesis inhibitor ketoconazole (KTC) on aspects of reproductive endocrine function controlled by the hypothalamic-pituitary-gonadal (HPG) axis in the fathead minnow (Pimephales promelas). Ketoconazole inhibits the activity of two cytochrome P450s (CYPs) key to sex steroid production in vertebrates, CYP11a (cholesterol side chain cleavage) and CYP17 (c17α-hydroxylase/17, 20-lyase). Sexually mature fish were exposed to water-borne KTC (30 or 300μg/L) in a flow-through system for up to 8d, following which animals were allowed to recover in clean water. Fish were sampled after 1, 4 and 8d of exposure, and after 1, 8 and 16d of recovery. A shorter-term time-course experiment also was conducted in which females were sampled on seven occasions during a 12h KTC exposure. Ketoconazole consistently depressed ex vivo gonadal synthesis of testosterone (T) in both sexes, and 17β-estradiol (E2) in females during both exposure and recovery phases of the time-course studies. Effects on ex vivo steroidogenesis in females occurred within as little as 1h of exposure. Plasma concentrations of T in males and E2 in females also were depressed by exposure to KTC, but these decreases did not persist to the same degree as observed for the ex vivo effects. In females, after decreases within 12h, plasma E2 concentrations were similar to (or greater than) controls at 24h of exposure, while in males, plasma T returned to levels comparable to controls within 1d of cessation of KTC exposure. The discrepancy between the ex vivo and in vivo data at later stages in the test is consistent with some type of compensatory response to KTC in fish. However, we were unable to ascertain the mechanistic basis for such a response. For example, although a number of genes related to steroid synthesis (e.g., cyp11a, cyp17) were up-regulated in the gonads of both males and females during the exposure and early recovery phases of the experiment, this did not seem to account for the resurgence in plasma steroid concentrations in KTC-exposed fish. Further studies focused on metabolism and clearance of steroids might lend insights as to the effects of KTC on plasma steroid concentrations. Overall, our results demonstrate the complex, temporally dynamic nature of the vertebrate HPG system in response to chemical stressors.