Systemic activation of glutamate dehydrogenase increases renal ammoniagenesis: implications for the hyperinsulinism/hyperammonemia syndrome.

The hyperinsulism/hyperammonemia (HI/HA) syndrome is caused by glutamate dehydrogenase (GDH) gain-of-function mutations that reduce the inhibition by GTP, consequently increasing the activity of GDH in vivo. The source of the hyperammonemia in the HI/HA syndrome remains unclear. We examined the effect of systemic activation of GDH on ammonia metabolism in the rat. 2-Aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) is a nonmetabolizable analog of the natural GDH allosteric activator leucine. A dose of 100 mumol BCH/100 g rat resulted in a mild systemic hyperammonemia. Using arterial-venous (A-V) differences, we exclude the liver, intestine, and skeletal muscle as major contributors to this BCH-induced hyperammonemia. However, renal ammonia output increased, as demonstrated by an increase in A-V difference for ammonia across the kidney in BCH-treated animals. Isolated renal cortical tubules incubated with BCH increased the rate of ammoniagenesis from glutamine by 40%. The flux through GDH increased more than twofold when BCH was added to renal mitochondria respiring on glutamine. The flux through glutaminase was not affected by BCH, whereas glutamate-oxaloacetate transaminase flux decreased when normalized to glutaminase flux. These data show that increased renal ammoniagenesis due to activation of GDH can explain the BCH-induced hyperammonemia. These results are discussed in relation to the organ source of the ammonia in the HI/HA syndrome as well as the role of GDH in regulating renal ammoniagenesis.

Reproductive toxicity of vinclozolin in the fathead minnow: confirming an anti-androgenic mode of action.

The objective of the present study was to characterize responses of the reproductive endocrine system of the fathead minnow (Pimephales promelas) to the fungicide vinclozolin (VZ), using a 21-d reproduction assay, and a shorter-term (approximately two weeks) test in which fish were cotreated with the VZ (a putative anti-androgen) and the androgen 17beta-trenbolone (TB). Effects on fecundity, gonadal histology, secondary sexual characteristics, reproductive hormones, and relative abundance of androgen receptor (AR) and 11beta-hydroxysteroid dehydrogenase (11betaHSD) mRNA transcripts were evaluated in one or both of these studies. Fecundity of VZ-exposed fish was decreased in a concentration-dependent manner in the 21-d test, culminating in complete reproductive failure at a concentration of 700 microg/L. Exposure to VZ decreased expression of male secondary sexual characteristics -- an effect typical of anti-androgens. The finding that exposure of females to TB-induced expression of prominent, male-like tubercles, which could be effectively blocked with VZ, provides powerful evidence of the anti-androgenic activity of VZ in vivo. In the two experiments VZ produced several responses possibly indicative of compensation or adaptation of the fish to the anti-androgen, including increases in gonad weight, AR and 11 betaHSD mRNA transcript abundance, and ex vivo gonadal production of testosterone and 11-ketotestosterone. Overall, our results demonstrate that the model anti-androgen VZ, which also is an environmental contaminant, impairs reproductive success of fathead minnows and elicits endocrine responses consistent with an anti-androgenic mode of action.

Ketoconazole in the fathead minnow (Pimephales promelas): reproductive toxicity and biological compensation.

Ketoconazole (KTC) is a model pharmaceutical representing imidazole and triazole pesticides, which inhibit fungal growth through blocking a cytochrome P450 (CYP)-mediated step in ergosterol biosynthesis. Several of these fungicides have been shown to be reversible inhibitors of CYPs in vertebrates (primarily mammals), including CYP isoforms involved in the pathway that converts cholesterol to active sex steroids. In these studies, we assessed the effects of KTC on aspects of steroidogenesis and reproductive function in the fathead minnow (Pimephales promelas). Exposure of spawning adults to the fungicide for 21 d significantly decreased egg production at a water concentration as low as 25 microg/L. Despite evidence of reduced ex vivo testosterone production by gonads from KTC-exposed fathead minnows, circulating plasma concentrations of sex steroids (testosterone, 17beta-estradiol) were not affected. Exposure to KTC caused an increase in the gonadosomatic index in both sexes and, in males, the fungicide caused a marked proliferation of interstitial (Leydig) cells. In addition, mRNA transcripts for two key steroidogenic enzymes, cytochrome P450 side-chain cleavage (CYP11A) and cytochrome P450 c17alpha hydroxylase/17,20 lyase (CYP17), were elevated by exposure to KTC. Both the changes in transcript levels and proliferation of gonad tissue represent potential adaptive or compensatory responses to impaired steroidogenic capacity. Overall our data indicate that, although KTC does adversely affect steroidogenesis and reproduction in the fathead minnow, the fish can compensate to some degree to mitigate effects of the fungicide. This has important implications for the interpretation of data from tests with endocrine-active chemicals.

Transcription of key genes regulating gonadal steroidogenesis in control and ketoconazole- or vinclozolin-exposed fathead minnows.

This study evaluated changes in the expression of steroidogenesis-related genes in male fathead minnows exposed to ketoconazole (KTC) or vinclozolin (VZ) for 21 days. The aim was to evaluate links between molecular changes and higher level outcomes after exposure to endocrine-active chemicals (EACs) with different modes of action. To aid our analysis and interpretation of EAC-related effects, we first examined variation in the relative abundance of steroidogenesis-related gene transcripts in the gonads of male and female fathead minnows as a function of age, gonad development, and spawning status, independent of EAC exposure. Gonadal expression of several genes varied with age and/or gonadal somatic index in either males or females. However, with the exception of aromatase, steroidogenesis-related gene expression did not vary with spawning status. Following the baseline experiments, expression of the selected genes in male fathead minnows exposed to KTC or VZ was evaluated in the context of effects observed at higher levels of organization. Exposure to KTC elicited changes in gene transcription that were consistent with an apparent compensatory response to the chemical's anticipated direct inhibition of steroidogenic enzyme activity. Exposure to VZ, an antiandrogen expected to indirectly impact steroidogenesis, increased pituitary expression of follicle-stimulating hormone beta-subunit as well as testis expression of 20beta-hydroxysteroid dehydrogenase and luteinizing hormone receptor transcripts. Results of this study contribute to ongoing research aimed at understanding responses of the teleost hypothalamic-pituitary-gonadal axis to different types of EACs and how changes in molecular endpoints translate into apical outcomes reflective of either adverse effect or compensation.

Comparison of fathead minnow ovary explant and H295R cell-based steroidogenesis assays for identifying endocrine-active chemicals.

An in vitro steroidogenesis assay using H295R human adenocarcinoma cells has been suggested as a possible alternative to gonad explant assays for use as a Tier I screening assay to detect endocrine active chemicals capable of modulating steroid hormone synthesis. This study is one of the first to investigate the utility of the H295R assay for predicting effects and/or understanding mechanisms of action across species and tissues. Six chemicals, including one selective aromatase inhibitor (fadrozole), four fungicides (fenarimol, ketoconazole, prochloraz, and vinclozolin), and one herbicide (prometon), were tested in both the H295R steroidogenesis assay, and an in vitro steroidogenesis assay using fathead minnow ovary explants. All six chemicals caused significant alterations in 17beta-estradiol (E2) and/or testosterone (T) production in vitro. Effects of ketoconazole, prochloraz, and prometon were similar in both assays. However, there were differences in the profile of responses for T for fadrozole and fenarimol, and for T and E2 for vinclozolin. In terms of sensitivity, steroid production in the H295R assay was most sensitive for detecting the effects of fadrozole, fenarimol, and prochloraz, but was less sensitive than the fathead minnow ovary explant assay to the effects of ketoconazole and vinclozolin. The H295R assay was consistently less variable (among replicates) than the fathead minnow ovary explant assay. However, the ovary explant assay was more predictive of in vivo effects of the six chemicals on fathead minnows than the H295R system. Further characterization of autoregulatory capacities, interaction of steroid-hormone receptor pathways with steroidogenesis, and metabolic capabilities of each system are needed for either system to provide clear and informative insights regarding a chemical's mechanism of action. Overall, however, results of this study suggest that both the H295R and fathead minnow ovary explant assays have utility for identifying endocrine-active chemicals in screening-type applications.

Development of quantitative real-time PCR assays for fathead minnow (Pimephales promelas) gonadotropin beta subunit mRNAs to support endocrine disruptor research.

Fathead minnows (Pimephales promelas) are a widely-used small fish model for regulatory ecotoxicology testing and research related to endocrine disrupting chemicals (EDCs). Quantitative real-time PCR assays for measuring fathead minnow gonadotropin (GtH) beta subunit transcripts were developed and "baseline" transcript levels in pituitary tissue were examined over a range of age classes and spawning states. Among females, GtHbeta transcripts did not vary significantly with gonadal-somatic index or gonad stage. However, in males, follicle-stimulating hormone beta subunit transcripts decreased significantly with increasing gonad stage, while mean luteinizing hormone beta subunit expression trended in the opposite direction. GtHbeta transcript levels measured in pituitaries from fish that had spawned within the preceding 24 h were not significantly different from those from fish that were 2-3 days post-spawn. Exposure to the fungicide ketoconazole, a known steroidogenesis inhibitor, for 21 days significantly affected the abundance of GtHbeta transcripts in pituitary tissue in males, but not females. This study provides critical data needed to design and interpret effective experiments for studying direct and indirect effects of EDCs on GtH subunit mRNA expression. Results of such experiments should facilitate a greater understanding of integrated system-wide responses of the fathead minnow brain-pituitary-gonadal axis to stressors including EDCs.

Relationship between brain and ovary aromatase activity and isoform-specific aromatase mRNA expression in the fathead minnow (Pimephales promelas).

There is growing evidence that some chemicals present in the environment have the capacity to inhibit, or potentially induce, aromatase activity. This study compared aromatase activities and isoform-specific mRNA expression in brain and ovary tissue from non-exposed fathead minnows representing three different ages and stages of reproductive activity, and from fathead minnows exposed to the aromatase inhibitor fadrozole for 7d. The goal was to determine whether measures of a single aromatase endpoint in either brain or ovary tissue would be sufficient to understand and predict system-wide effects of endocrine disrupting chemicals on aromatase activity and transcript levels. Aromatase activity in the ovary, but not brain, varied significantly with age/reproductive category, with adults held in non-reproductive conditions showing significantly lower activity than juveniles and reproductively-active adults. Significant correlations between isoform-specific transcript levels and aromatase activity were observed for ovary tissue, but those relationships were not robust for all age/reproductive categories, nor were they sustained in fadrozole-treated fish. In vitro, fadrozole inhibited the aromatase activity of brain and ovary post-mitochondrial supernatants with similar potency (IC50s = 8.82 +/- 1.58 and 6.93 +/- 0.80 microM for brain and ovary, respectively), despite large differences in the magnitude of activity. In vivo, fadrozole altered aromatase activity and isoform-specific transcript levels in both brain and ovary tissue, but concentration-response relationships were different for each tissue. Aromatase activity and P450aromB mRNA expression in brain showed a dose-dependent decrease at concentrations greater than 5.55 microg/L. In contrast, ovary activity showed an inverted U-shaped concentration-response consistent with the interplay between increased P450aromA transcript levels in ovary and competitive inhibition of the aromatase enzyme. As a whole, results of this study did not reveal any robust correlations between brain and ovary aromatase activity and/or isoform-specific mRNA expression. However, they were consistent with the current body of evidence related to teleost aromatase regulation, suggesting that increased understanding of the biology of aromatase may facilitate system-wide understanding of effects on aromatase based on relatively few measured endpoints.