Physiological and molecular responses to environmental pollutants in Atlantic cod (Gadus morhua) : Characterization of the aryl hydrocarbon receptor signaling pathway and underlying mechanisms of crude oil toxicity

Abstract

Environmental pollutants are harmful substances that can interfere and disrupt numerous physiological processes. Different cellular defence mechanisms are therefore crucial for both detecting pollutants and coordinate the transcription and synthesis of genes and enzymes that are able to metabolize and excrete such compounds from the cells. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor with an important role in mediating xenobiotic responses to a variety of environmental pollutants in vertebrates, including fish. The marine environment is the ultimate sink for environmental pollutants, and Atlantic cod (Gadus morhua) has been used as a bioindicator species to monitor the effects of marine pollution during the last decades. Atlantic cod is also an economically and ecologically important species in the North Atlantic Ocean, whose important spawning grounds are located in areas with proposed offshore oil exploration activities in Norway.

In Paper I, the molecular and functional properties of the Atlantic cod Ahr1a and Ahr2a paralogs were characterized. In vitro binding affinity and transactivation studies showed that the cod Ahrs can bind and be activated by several mammalian AHR agonists, but Ahr1a demonstrated the greatest affinity and sensitivity towards 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Tissue-specific expression of ahr1a, ahr2a, arnt1 and arnt2 was assessed in juvenile cod, and ahr2a and arnt1 were the most abundantly expressed genes across the different tissues examined, including liver. The induction of the Ahr signaling pathway was also confirmed ex vivo using cod liver slices exposed to different AHR ligands, including 3,3´,4,4´,5-pentachlorobiphenyl (PCB126), 6-formylindolo[3,2-b]carbazole (FICZ) and benzo[a]pyrene (B[a]P), where induction of cyp1a expression was observed for all of these compounds. Based on the abundant expression in cod liver, it was suggested that Ahr2a is the most likely protein to be involved in mediating responses to xenobiotic exposure, but a possible role of Ahr1a should not be excluded. Furthermore, the different binding affinities and sensitivities, as well as the tissue-specific expression profiles of Ahr1 and Ahr2a, indicated that a subfunctional partitioning of the cod Ahrs has occurred.

Paper II focused on further exploring the possible subfunctional partitioning of Ahr1a and Ahr2a during early life stages (ELS) of Atlantic cod. Activation of the Ahr signaling pathway in embryos exposed to B[a]P was demonstrated, and expression and localization of ahr1a, ahr2a and cyp1a were assessed with in situ hybridization. Induced expression of ahr2a and cyp1a was observed in the cardiovascular system and skin, respectively, of B[a]P-exposed cod embryos and larvae. Furthermore, expression of ahr2a and cyp1a was also evident in the liver of B[a]P-exposed larvae. Our results further supported that a subfunctional partitioning of the ahr1a and ah2a paralogs has occurred, and that Ahr2a is the major subtype involved in mediating xenobiotic responses during ELS of Atlantic cod. On the other hand, ahr1a showed a persistent expression in the eye of cod embryos and larvae independent of B[a]P exposure, suggesting a role of Ahr1a in the development of the eye during early development. An endogenous role of Ahr2a was also indicated by its expression in the jaws and fin nodes of larvae.

In Paper III, a possible photo-enhanced toxicity of crude oil to ELS of Atlantic cod was studied. Several marine fish species, including Atlantic cod, have their spawning and nursery grounds in areas along the coast of Northern Norway, where offshore oil activities have been proposed. The toxicity of crude oil is known to be enhanced by exposure to ultraviolet (UV) radiation present in sunlight. Cod embryos and larvae are transparent and pelagic, and in the possible event of an oil spill during spawning in the spring months, they could experience photo-enhanced toxicity of crude oil. Atlantic cod embryos exposed to crude oil with the presence of UV radiation demonstrated increased mortality at high crude oil doses in comparison to embryos exposed to crude oil alone. Phenotypic outcomes, such as craniofacial malformations and heart deformities were visible in larvae from both experiments at the highest crude oil doses, whereas spinal curvature deformities were predominant in larvae exposed to crude oil with UV radiation. Higher number of differentially expressed genes (DEGs) and enriched pathways were revealed in co-exposed embryos. Interaction analyses revealed that several of the enriched pathways were affected by the co-treatment with UV radiation, including circadian rhythm, oxidative stress, mitochondrial function, tryptophan metabolism and retinoid metabolism, indicating a photo-enhanced effect of crude oil toxicity on these physiological and cellular processes. Phenotypic traits observed in larvae appear to be manifested in the transcriptome responses affected by the treatments, providing mechanistic insights into crude oil and photo-enhanced crude oil toxicity. Our results suggest that UV radiation increases the toxicity of crude oil in ELS of Atlantic cod.