Mathilakath M. Vijayan

Cortisol in teleosts: dynamics, mechanisms of action, and metabolic regulation

Cortisol is the principal corticosteriod in teleost fishes and its plasma concentrations rise dramatically during stress. The relationship between this cortisol increase and its metabolic consequences are subject to extensive debate. Much of this debate arises from the different responses of the many species used, the diversity of approaches to manipulate cortisol levels, and the sampling techniques and duration. Given the extreme differences in experimental approach, it is not surprising that inconsistencies exist within the literature. This review attempts to delineate common themes on the physiological and metabolic roles of cortisol in teleost fishes and to suggest new approaches that might overcome some of the inconsistencies on the role of this multifaceted hormone. We detail the dynamics of cortisol, especially the exogenous and endogenous factors modulating production, clearance and tissue availability of the hormone. We focus on the mechanisms of action, the biochemical and physiological impact, and the interaction with other hormones so as to provide a conceptual framework for cortisol under resting and/or stressed states. Interpretation of interactions between cortisol and other glucoregulatory hormones is hampered by the absence of adequate hormone quantification, resulting in correlative rather than causal relationships.The use of mammalian paradigms to explain the teleost situation is generally inappropriate. The absence of a unique mineralocorticoid and likely minor importance of glucose in fishes means that cortisol serves both glucocorticoid and mineralocorticoid roles; the unusual structure of the fish glucocorticoid receptor may be a direct consequence of this duality. Cortisol affects the metabolism of carbohydrates, protein and lipid. Generally cortisol is hyperglycaemic, primarily as a result of increases in hepatic gluconeogenesis initiated as a result of peripheral proteolysis. The increased plasma fatty acid levels during hypercortisolaemia may assist to fuel the enhanced metabolic rates noted for a number of fish species. Cortisol is an essential component of the stress response in fish, but also plays a significant role in osmoregulation, growth and reproduction. Interactions between cortisol and toxicants may be the key to the physiology of this hormone, although cortisols many important housekeeping functions must not be ignored. Combining molecular approaches with isolated cell systems and the whole fish will lead to an improved understanding of the many faces of this complex hormone in an evolutionary and environmental framework.

Exposure and effects assessment of persistent organohalogen contaminants in arctic wildlife and fish

Persistent organic pollutants (POPs) encompass an array of anthropogenic organic and elemental substances and their degradation and metabolic byproducts that have been found in the tissues of exposed animals, especially POPs categorized as organohalogen contaminants (OHCs). OHCs have been of concern in the circumpolar arctic for decades. For example, as a consequence of bioaccumulation and in some cases biomagnification of legacy (e.g., chlorinated PCBs, DDTs and CHLs) and emerging (e.g., brominated flame retardants (BFRs) and in particular polybrominated diphenyl ethers (PBDEs) and perfluorinated compounds (PFCs) including perfluorooctane sulfonate (PFOS) and perfluorooctanic acid (PFOA) found in Arctic biota and humans. Of high concern are the potential biological effects of these contaminants in exposed Arctic wildlife and fish. As concluded in the last review in 2004 for the Arctic Monitoring and Assessment Program (AMAP) on the effects of POPs in Arctic wildlife, prior to 1997, biological effects data were minimal and insufficient at any level of biological organization. The present review summarizes recent studies on biological effects in relation to OHC exposure, and attempts to assess known tissue/body compartment concentration data in the context of possible threshold levels of effects to evaluate the risks. This review concentrates mainly on post-2002, new OHC effects data in Arctic wildlife and fish, and is largely based on recently available effects data for populations of several top trophic level species, including seabirds (e.g., glaucous gull (Larus hyperboreus)), polar bears (Ursus maritimus), polar (Arctic) fox (Vulpes lagopus), and Arctic charr (Salvelinus alpinus), as well as semi-captive studies on sled dogs (Canis familiaris). Regardless, there remains a dearth of data on true contaminant exposure, cause-effect relationships with respect to these contaminant exposures in Arctic wildlife and fish. Indications of exposure effects are largely based on correlations between biomarker endpoints (e.g., biochemical processes related to the immune and endocrine system, pathological changes in tissues and reproduction and development) and tissue residue levels of OHCs (e.g., PCBs, DDTs, CHLs, PBDEs and in a few cases perfluorinated carboxylic acids (PFCAs) and perfluorinated sulfonates (PFSAs)). Some exceptions include semi-field studies on comparative contaminant effects of control and exposed cohorts of captive Greenland sled dogs, and performance studies mimicking environmentally relevant PCB concentrations in Arctic charr. Recent tissue concentrations in several arctic marine mammal species and populations exceed a general threshold level of concern of 1 part-per-million (ppm), but a clear evidence of a POP/OHC-related stress in these populations remains to be confirmed. There remains minimal evidence that OHCs are having widespread effects on the health of Arctic organisms, with the possible exception of East Greenland and Svalbard polar bears and Svalbard glaucous gulls. However, the true (if any real) effects of POPs in Arctic wildlife have to be put into the context of other environmental, ecological and physiological stressors (both anthropogenic and natural) that render an overall complex picture. For instance, seasonal changes in food intake and corresponding cycles of fattening and emaciation seen in Arctic animals can modify contaminant tissue distribution and toxicokinetics (contaminant deposition, metabolism and depuration). Also, other factors, including impact of climate change (seasonal ice and temperature changes, and connection to food web changes, nutrition, etc. in exposed biota), disease, species invasion and the connection to disease resistance will impact toxicant exposure. Overall, further research and better understanding of POP/OHC impact on animal performance in Arctic biota are recommended. Regardless, it could be argued that Arctic wildlife and fish at the highest potential risk of POP/OHC exposure and mediated effects are East Greenland, Svalbard and (West and South) Hudson Bay polar bears, Alaskan and Northern Norway killer whales, several species of gulls and other seabirds from the Svalbard area, Northern Norway, East Greenland, the Kara Sea and/or the Canadian central high Arctic, East Greenland ringed seal and a few populations of Arctic charr and Greenland shark.

Cortisol treatment affects glucocorticoid receptor and glucocorticoid-responsive genes in the liver of rainbow trout.

We investigated whether longer-term cortisol exposure modified hepatic glucocorticoid receptor (GR) status and tissue responsiveness to cortisol stimulation in rainbow trout. Fish were given intraperitoneal implants of cortisol (50mg/kg body mass) and this led to elevated plasma cortisol levels mimicking chronically stressed salmonids. There was significantly higher hepatic GR mRNA abundance, despite a drop in GR protein content in the liver of cortisol-treated fish. The tissue responsiveness to cortisol stimulation was apparent from the higher plasma glucose concentration and liver glycogen content. Also, the higher phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance, a key glucocorticoid-responsive gene, by cortisol suggests activation of the GR signalling pathway. There was no significant effect of cortisol treatment on liver PEPCK, alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase activities compared to the sham fish. The higher heat shock protein (hsp) 90 mRNA abundance and a corresponding elevation in this protein and constitutive hsp70 (hsc70) protein content in the cortisol-treated fish reflects a role for glucocorticoids in the hepatic stress response process. Taken together, the molecular and biochemical responses evident in the liver of trout imply changes favouring tissue responsiveness to glucocorticoids and may be a mechanism to offset GR protein downregulation evident with chronic cortisol stimulation in rainbow trout.

Stress transcriptomics in fish: A role for genomic cortisol signaling ☆

The physiological responses to stressors, including hormonal profiles and associated tissue responsiveness have been extensively studied in teleosts, but the molecular mechanisms associated with this adaptive response are not well understood. The advent of cDNA microarray technology has transformed the field of functional genomics by revealing global gene expression changes in response to stressor exposures even in non-mammalian vertebrates, including fish. A unifying response in studies related to stressor exposure is activation of the hypothalamus-pituitary-interrenal (HPI) axis in fish, leading to cortisol release into the circulation. Here we will discuss the implications of some of the gene expression changes observed in response to acute stress in fish, while highlighting a role for cortisol in this adaptive stress response. As liver is a key organ for metabolic adjustments to stressors and also is a major target for cortisol action, the genomic studies pertaining to stress and glucocorticoid regulation have focused mainly on this tissue. The studies have identified several genes that are altered transiently after an acute stressor exposure in fish. A number of these stress-responsive genes were also modulated by glucocorticoid receptor activation, suggesting that elevation in cortisol levels during stressor exposure may be a key signal for target tissue molecular programming, essential for stress adaptation. The identification of regulatory gene networks that are stress activated, and modulated by cortisol, both in hepatic and extra-hepatic tissues, including gonads, brain, immune cells and gills, will provide a mechanistic framework to characterize the multifaceted role of cortisol during stress adaptation.

The zebrafish stress axis: molecular fallout from the teleost-specific genome duplication event.

The teleost-specific whole genome duplication event 350 million years ago resulted in a variety of duplicated genes that exist in fish today. In this review, we examine whether molecular components involved in the functioning of the hypothalamus-pituitary-interrenal (HPI) axis are present as single or duplicate genes. Specifically, we looked at corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and the glucocorticoid receptor (GR). The focus is on zebrafish but a variety of species are covered whenever data is available through literature or genomic database searches. Duplicate CRH genes are retained in the salmoniformes and cypriniformes, and the peptide sequences are very similar or identical. Zebrafish, along with the Acanthopterygii, are the exceptions as they have a single CRH gene. Also, two copies of the proopiomelanocortin (POMC) gene, which encodes for ACTH and other peptides, have been observed in all teleosts except tilapia and sea bass. In zebrafish, ACTH is derived from only one POMC gene, since the cleavage site is mutated in the other gene. All teleosts examined to date have two GRs, including the recent discoveries of duplicate GRs in two species of cyprinids (carp and fathead minnow). Zebrafish are the only known exception with one GR gene. The loss of duplicate genes is not a general feature of the zebrafish genome, but zebrafish have lost the duplicate CRH, ACTH and GR genes in the past 33 million years, after possessing two of each for the previous 300 million years. The evolutionary pressures underlying the rapid loss of these HPI axis genes, and the implications on the development and the functioning of the evolutionarily conserved cortisol stress response in zebrafish are currently unknown.

Molecular programming of the corticosteroid stress axis during zebrafish development.

The functions of the hypothalamus-pituitary-interrenal (HPI) axis in teleosts have been studied primarily in juvenile and adult fish, whereas little is known about the molecular events leading to the onset of the stressor-induced cortisol response during development. Here we summarize a number of studies that have examined changes in the expression of genes encoding proteins critical for the functioning of the HPI axis, and the associated cortisol response in developing zebrafish embryos and larvae. The mRNA transcripts for some of these genes, including corticotropin releasing factor (CRF), proopiomelanocortin (POMC), melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage (P450scc) have been detected during embryogenesis prior to hatch. The mRNA levels of MC2R, StAR and P450scc are up-regulated immediately prior to the dramatic rise in basal larval cortisol levels after hatch. Although all the components of the HPI axis are expressed and cortisol is synthesized at hatch, a stressor-induced cortisol response was not evident until 97 hpf. We hypothesize that this disconnect in the timing of the basal cortisol synthesis and stressor-induced cortisol synthesis is due to the delayed development of peripheral and central neural inputs relaying stressor stimuli to the hypothalamus. Overall, zebrafish appear to be an excellent model for elucidating the molecular mechanisms leading to the development of the corticoid stress axis in vertebrates.

Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure.

The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells, whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout hepatocytes subjected to a heat shock (+15 degrees C for 1 h) or exposed to either CuSO(4) (200 microM for 24 h), CdCl(2) (10 microM for 24 h) or NaAsO(2) (50 microM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70 content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular stress response process in fish.

Gene expression pattern in the liver during recovery from an acute stressor in rainbow trout.

The physiological response to stressors, including hormonal profiles and associated tissue responsiveness, has been extensively studied with salmonid fish, but less is known about the molecular basis of this adaptive response. As liver is the major target organ for metabolic adjustments, we exploited a selective transcriptomics approach to address molecular response in this tissue during acute stress adaptation in rainbow trout. The stressor consisted of a standardized 3 min handling disturbance of trout, and plasma and liver samples were collected either prior to or 1 and 24 h after stressor exposure. We developed a low density custom cDNA array consisting of 147 rainbow trout genes designed specifically to represent stress-responsive and endocrine-related pathways in fish. The acute stress response and recovery was confirmed by the transient elevation in plasma cortisol concentration at 1 h, which returned to pre-stress levels over a 24 h period. This was accompanied by significant upregulation of 40 genes at 1 h, and 15 genes at 24 h after stressor exposure in trout liver. Many of these genes were involved in energy metabolism, implicating a rapid liver molecular reprogramming as critical for the metabolic adjustments to an acute stressor. Several other transcripts not previously implicated in the stress response process in fish, including genes involved in immune function and protein degradative pathways, were found to be stress-responsive in trout. A large number of these stress-responsive transcripts were also shown previously to be glucocorticoid-responsive in fish. Together, our results suggest a role for stressor-mediated genomic cortisol signaling in the liver molecular programming associated with stress in fish. Overall, the study demonstrates the complex nature of the adaptive stress response at the molecular level and underscores the utility of targeted gene expression studies for identifying stress coping mechanisms.

Cortisol modulates HSP90 mRNA expression in primary cultures of trout hepatocytes.

The objective of the present study was to understand the role of cortisol in the cellular stress response process in fish. Specifically, our studies addressed whether cortisol exposure modified heat shock protein 90 (HSP90) mRNA expression in rainbow trout (Oncorhynchus mykiss) hepatocytes maintained in primary culture. We also subjected these hepatocytes to heat shock (HS) in order to examine the role of cortisol on HS-induced HSP90 mRNA expression. A cDNA fragment of 500 bp was cloned from trout liver by reverse transcriptase- polymerase chain reaction (RT-PCR) with primers designed from the conserved regions of chinook salmon and zebrafish HSP90 cDNAs. The PCR product showed very high homology to chinook salmon (98%), zebrafish (84%) and human (77%) HSP90. Heat shock (+6 degrees C) induced transient elevation in HSP90 mRNA in trout hepatocytes, peaking within 10-h post-HS, and remained elevated over a 24-h period. Cortisol did not modify the unstimulated expression of HSP90 mRNA, whereas the HS-induced HSP90 mRNA expression was attenuated in trout hepatocytes. Our results suggest that elevated plasma cortisol levels modulate the cellular stress response by affecting the transcription of HSP90 in fish.

Central and peripheral glucocorticoid receptors are involved in the plasma cortisol response to an acute stressor in rainbow trout.

Cortisol, the primary circulating corticosteroid in teleosts, is elevated during stress following activation of the hypothalamus-pituitary-interrenal (HPI) axis. Cortisol exerts genomic effects on target tissues in part by activating glucocorticoid receptors (GR). Despite a well-established negative feedback loop involved in plasma cortisol regulation, the role of GR in the functioning of the HPI axis during stress in fish is still unclear. We used mifepristone (a GR antagonist) to suppress GR signaling in rainbow trout (Oncorhynchus mykiss) and assessed the resultant changes to HPI axis activity. We show for the first time that mifepristone caused a functional knockdown of GR by depleting protein expression 40-75%. The lower GR protein expression corresponded with a compensatory up-regulation of GR mRNA levels across tissues. Mifepristone treatment completely abolished the stressor-induced elevation in plasma cortisol and glucose levels seen in the control fish. A reduction in corticotropin-releasing factor (CRF) mRNA abundance in the hypothalamic preoptic area was also observed, suggesting that GR signaling is involved in maintaining basal CRF levels. We further characterized the effect of mifepristone treatment on the steroidogenic capacity of interrenal tissue in vitro. A marked reduction in cortisol production following adrenocorticotropic hormone stimulation of head kidney pieces was observed from mifepristone treated fish. This coincided with the suppression of steroidogenic acute regulatory protein, but not P450 side chain cleavage mRNA abundances. Overall, our results underscore a critical role for central and peripheral GR signaling in the regulation of plasma cortisol levels during stress in fish.