Oxidative Stress Parameters in Goitrogen-Exposed Crested Newt Larvae (Triturus spp.): Arrested Metamorphosis

Thiourea is an established disruptor of thyroid hormone synthesis and is frequently used as an inhibitor of metamorphosis. The changes caused by thiourea can affect processes associated with the oxidative status of individuals (metabolic rate, the HPI axis, antioxidant system). We investigated the parameters of oxidative stress in crested newt (Triturus spp.) larvae during normal development in late larval stage 62 and newly metamorphosed individuals, and during thiourea-stimulated metamorphosis arrest in individuals exposed to low (0.05%) and high (0.1%) concentrations of thiourea. Both groups of crested newts exposed to thiourea retained their larval characteristics until the end of the experiment. The low activities of antioxidant enzymes and the high lipid peroxidation level pointed to increased oxidative stress in larvae at the beginning of stage 62 as compared to fully metamorphosed individuals. The activities of catalase (CAT) and glutathione-S-transferase (GST) and the concentration of sulfhydryl (SH) groups were significantly lower in larvae reared in aqueous solutions containing thiourea than in newly metamorphosed individuals. The high thiourea concentration (0.1%) affected the antioxidative parameters to the extent that oxidative damage could not be avoided, contrary to a lower concentration. Our results provide a first insight into the physiological adaptations of crested newts during normal development and simulated metamorphosis arrest.

Persistent Exposure to Environmental Levels of Microcystin-LR Disturbs Cortisol Production via Hypothalamic-Pituitary-Interrenal (HPI) Axis and Subsequently Liver Glucose Metabolism in Adult Male Zebrafish (Danio rerio)

There is growing evidence that microcystin-LR (MC-LR) is a new endocrine disruptor, whereas the impacts of persistent exposure to MC-LR on the hypothalamic-pituitary-interrenal (HPI) axis and health hazards thereafter have not been investigated. In this work, adult male zebrafish (Danio rerio) were immersed into MC-LR solutions at concentrations of 0, 1, 5 and 25 μg/L for 30 d, respectively. The results showed that persistent MC-LR exposure caused an extensive upregulation of HPI-axis genes but an inhibition of brain nuclear receptors (gr and mr), which finally increased serum cortisol levels. Furthermore, the decreased expression of hepatic gr might partly be responsible for the strong inhibition on the expression of downstream genes involved in glucose metabolic enzymes, including gluconeogenesis-related genes (pepck, fbp1a, g6pca), glycogenolysis-related gene (pyg), glycolysis-related genes (gk, pfk1b, pk) and glycogenesis-related gene (gys2). These findings are in accordance with the decline in serum glucose, indicating that long-term MC-LR exposure caused a lower production of glucose relative to glucose lysis. Our above results firstly establish the link between persistent MC-LR exposure and impaired glucose metabolism, suggesting that long-term MC-LR-mediated stress might threaten fish’s health.

Effects of early life glucocorticoid exposure on metabolism in zebrafish (Danio rerio) larvae

In this study we assessed the effects of increased cortisol levels during early embryonic development (0-6 hours post-fertilisation (hpf)), thereby mimicking maternal stress, on metabolism in zebrafish (Danio rerio) larvae. In two series of experiments fertilized eggs were exposed to a cortisol-containing, a dexamethasone-containing (to stimulate the glucocorticoid receptor (GR) specifically) or a control medium for 6 hours post-fertilisation (0-6 hpf). In the first series we measured oxygen consumption as a proxy for metabolism, in the second series gene-expression of genes related to gluconeogenesis and glucose transport. Previously we have found that at 5 days post-fertilisation (dpf) baseline cortisol levels are increased following cortisol pre-treatment but not following dexamethasone pre-treatment, suggesting a higher hypothalamus-pituitary-interrenal cells (HPI-axis) activity. Hence, we hypothesized that oxygen consumption and gene-expression were stronger in cortisol-treated than in dexamethasone-treated and control-treated subjects at 5 dpf. Indeed, we observed increased oxygen consumption in cortisol-treated subjects compared to dexamethasone-treated or control-treated subjects. However, gene-expression levels were not different between treatments, which may have been due to a developmental delay in this second series. We also reasoned that both cortisol-treated and dexamethasone-treated subjects would show a higher metabolism at 1 dpf than control-treated subjects as the HPI-axis is not functional as yet and more general processes are being stimulated by cortisol through GR stimulation. Indeed, we observed increased oxygen consumption and increased expression of genes related to gluconeogenesis and glucose transport in cortisol-treated and dexamethasone-treated subjects than control-treated subjects. These data show that early-life exposure to cortisol, mimicking thereby maternal stress, increased metabolism at different life stages, i.e. 1 and 5 dpf, involving the GR.

Locomotor response to acute stressors requires hypothalamic-pituitary-interrenal axis activation and glucocorticoid receptor

When vertebrates face acute stressors, their bodies rapidly undergo a repertoire of physiological and behavioral adaptations, which is termed the stress response (SR). Rapid physiological changes in heart rate and blood sugar levels occur via the interaction of glucocorticoids and their cognate receptors following hypothalamic-pituitary-adrenal (HPA) axis activation. These physiological changes are observed within minutes of encountering a stressor and the rapid time domain rules out genomic responses that require gene expression changes. Although behavioral changes corresponding to physiological changes are commonly observed, it is not clearly understood to what extent HPA axis activation dictates adaptive behavior. We hypothesized that rapid locomotor response to acute stressors in zebrafish requires HPI axis activation. In teleost fish, interrenal cells (I) are functionally homologous to the adrenal gland cortical layer. We derived 8 frameshift mutants in genes involved in HPI axis function: two mutants in exon 2 of mc2r (adrenocorticotropic hormone receptor), two in each of exon 2 and exon 5 of nr3c1 (glucocorticoid receptor), and two in exon 2 of nr3c2 (mineralocorticoid receptor). Exposing larval zebrafish to mild environmental stressors, acute changes in salinity or light illumination, results in a rapid locomotor response. We show here that this locomotor response requires a functioning HPI axis via the action of mc2r (adrenocorticotropic hormone receptor) and the canonical glucocorticoid receptor encoded by nr3c1 gene, but not mineralocorticoid receptor (nr3c2). Our rapid behavioral assay paradigm based on HPI axis biology may prove useful to screen for genetic, pharmacological, or environmental modifiers of the HPA axis.SignificanceAltered HPA axis activity is acknowledged as a causative and critical prognostic factor in many psychiatric disorders including depression. Nonetheless, genome wide association studies (GWAS) on depression have revealed conflicting findings about susceptibility loci, while identifying several genetic loci that warrant further investigations in the process. Such findings indicate that psychiatric disorders with complex genetic foundations require functional studies as well as genetic analyses. We developed a sensitive behavioral assay paradigm that leverages the genetic amenability and rapid development of zebrafish and demonstrated that our assay system reliably detects changes in HPA axis responsiveness. Our functional genetics and behavioral assay approach provides a useful platform to discover novel genetic, pharmacological, or environmental modifiers of the HPA axis.