Marit Nøst Hegseth

The interplay between infectious pancreatic necrosis virus (IPNV) and the IFN system: IFN signaling is inhibited by IPNV infection

Abstract Infectious pancreatic necrosis virus (IPNV) is a major pathogen in the aquaculture industry worldwide. Factors contributing to IPNV pathogenicity are yet poorly understood. Indications of IPNV being able to evade or counteract innate host defense come from its lack of ability to induce strong type I interferon (IFN) responses in cell culture. We show here that addition of salmon rIFN-α1 to cells prior to IPNV infection halts the viral protein synthesis and prevents processing of pVP2 into mature VP2. Furthermore, compared to pre-treatment with IFN-α1 the antiviral state in cells infected with IPNV prior to IFN-treatment, was antagonized by IPNV, as detected by higher viral titers, faster viral protein synthesis and also by reduced Mx expression. The longer headstart the virus gets, the more prominent is the weakening of IFN signaling. IPNV VP4 and VP5 inhibit IFN-induced expression from the Mx promoter, indicating that these proteins contribute to the antagonistic effect.

Biotransformation of PCBs in Arctic seabirds: Characterization of phase I and II pathways at transcriptional, translational and activity levels

Arctic seabirds are exposed to a wide range of halogenated organic contaminants (HOCs). Exposure occurs mainly through food intake, and many pollutants accumulate in lipid-rich tissues. Little is known about how HOCs are biotransformed in arctic seabirds. In this study, we characterized biotransformation enzymes in chicks of northern fulmars (Fulmarus glacialis) and black-legged kittiwakes (Rissa tridactyla) from Kongsfjorden (Svalbard, Norway). Phase I and II enzymes were analyzed at the transcriptional, translational and activity levels. For gene expression patterns, quantitative polymerase chain reactions (qPCR), using gene-sequence primers, were performed. Protein levels were analyzed using immunochemical assays of western blot with commercially available antibodies. Liver samples were analyzed for phase I and II enzyme activities using a variety of substrates including ethoxyresorufin (cytochrome (CYP)1A1/1A2), pentoxyresorufin (CYP2B), methoxyresorufin (CYP1A), benzyloxyresorufin (CYP3A), testosterone (CYP3A/CYP2B), 1-chloro-2,4-nitrobenzene (CDNB) (glutathione S-transferase (GST)) and 4-nitrophenol (uridine diphosphate glucuronyltransferase (UDPGT)). In addition, the hydroxylated (OH-) polychlorinated biphenyls (PCBs) were analyzed in the blood, liver and brain tissue, whereas the methylsulfone (MeSO(2)-) PCBs were analyzed in liver tissue. Results indicated the presence of phase I (CYP1A4/CYP1A5, CYP2B, and CYP3A) and phase II (GST and UDPGT) enzymes at the activity, protein and/or mRNA level in both species. Northern fulmar chicks had higher enzyme activity than black-legged kittiwake chicks. This in combination with the higher SigmaOH-PCB to parent PCB ratios suggests that northern fulmar chicks have a different biotransformation capacity than black-legged kittiwake chicks.

Hepatic antioxidant responses related to levels of PCBs and metals in chicks of three Arctic seabird species.

The efficiency of antioxidant defenses and relationship with body burden of metal and organic contaminants has not been previously investigated in arctic seabirds, neither in chicks nor in adults. The objective of this study was to compare such defenses in chicks from three species, Black-legged kittiwake (Rissa tridactyla), Northern fulmar (Fulmarus glacialis), and Herring gull (Larus argentatus), and the relationship with tissue concentrations of essential metals such as selenium and iron and halogenated organic compounds, represented by polychlorinated biphenyl (PCB). The results showed significant species-specific differences in the antioxidant responses which also corresponded with metal and PCB levels in different ways. The capability to neutralize hydroxyl radicals (TOSC-HO•) and the activities of catalase and Se-dependent glutathione peroxidases (GPX) clearly increased in species with the higher levels of metals and PCBs, while the opposite trend was observed for Se-independent GPX, TOSC against peroxyl radicals (ROO•) and peroxynitrite (ONOOH). Less clear relationships were obtained for glutathione levels, GSH/GSSG ratio, glutathione reductase and superoxide dismutase. The results showed differences in antioxidant efficiency between the species, and some of these defenses exhibited dose-response-like relationships with measured levels of selenium, iron and ΣPCBs. PCBs, selenium and iron levels were positively related to the responses of antioxidants with potential to reduce HO•/H₂O₂ (Se-dependent GPX, CAT and TOSC against HO•). However, direct causal relationships between antioxidant responses and contaminant concentrations could not be shown on individual level. Varying levels of metals and contaminants due to different diet and age were probably the main explanations for the species differences in antioxidant defense.

Effects of exposure to halogenated organic compounds combined with dietary restrictions on the antioxidant defense system in herring gull chicks

The Herring gull (HG) (Larus argentatus) is naturally exposed to halogenated organic compounds such as polychlorinated biphenyls (PCBs) through its diet. During periods of food scarcity, arctic seabirds experience lipid mobilization, allowing stored lipid soluble contaminants to re-enter the body circulation. In the present study, we investigated the effects of PCB exposure and fasting on the antioxidant defense system in HG chicks. Forty newly hatched chicks were exposed to contaminated cod liver oil for 6weeks and then fasted for 1week. We assessed the hepatic total oxyradical scavenging capacity (TOSC) against peroxynitrite, hydroxyl and peroxyl radicals, and measured glutathione (reduced: GSH, and oxidized: GSSG) levels and the enzymatic activities of catalase, glutathione peroxidase, glutathione reductase and superoxide dismutase. The results show that fasting significantly increased the HOC levels in the HG chick livers. Limited effects were observed on antioxidant responses; significant effects were only found for catalase (CAT) activity, Se-dependent GPX activity and the GSH/GSSG ratio in the exposed and fasted group. CAT and Se-dependent GPX activities correlated negatively with the PCB concentrations within this group, and a nonlinear relationship between glutathione and contaminant levels was also found. These effects were generally not observed after exposure or fasting alone and were likely related to the high PCB levels induced by the combination of exposure and fasting.

Lysosomal and lipid-associated parameters in the livers of three species of arctic seabird chicks: species differences and relationships with contaminant levels.

Lysosomal membrane stability, lipofuscin (LF), malondialdehyde (MDA), neutral lipid (NL) levels, as well as halogenated organic compounds (HOCs), Cr, Cd, Pb and Fe concentrations were analyzed in liver of black-legged kittiwake (BK), herring gull (HG), and northern fulmar (NF) chicks. There were significant species differences in the levels of NL, LF and lysosomal membrane stability. These parameters were not associated with the respective HOC concentrations. LF accumulation was associated with increasing Cr, Cd and Pb concentrations. HG presented the lowest lysosomal membrane stability and the highest. LF and NL levels, which indicated impaired lysosomes in HG compared to NF and BK. Lipid peroxidation was associated with HOC and Fe2+ levels. Specific HOCs showed positive and significant correlations with MDA levels in HG. The study indicates that contaminant exposure can affect lysosomal and lipid associated parameters in seabird chicks even at low exposure levels. These parameters may be suitable markers of contaminant induced stress in arctic seabirds.

Effects of contaminant exposure and food restriction on hepatic autophagic lysosomal parameters in Herring Gull (Larus argentatus) chicks

Lysosomal autophagic responses, such as lysosomal membrane stability, neutral lipids (NL), lipofuscin (LF), and malondialdehyde (MDA) levels, are valuable measures of cellular early-onset effects induced by environmental stress factors, such as contaminant exposure and fasting. In this study, these parameters were analysed and related to levels of halogenated organic contaminants (HOCs) in 40 Herring Gull (Larus argentatus) chicks. Chicks were experimentally exposed to HOCs through diet and went through a period of nutrient deprivation at the end of the experiment. HOC exposure and fasting were conducted separately and in combination. NL storages were depleted, and lysosomal membranes were destabilised after HOC exposure and nutrient deprivation. These responses were not related specifically to one type of stress or the extent of the treatment. No synergistic or additive effects from the combination of HOC exposure and fasting were observed. LF accumulated, and MDA levels increased as a result of fasting, but were unaffected by HOC exposure. LF accumulation was strongly associated with the percent weight change in the chicks. Large weight loss was associated with high LF levels, and slight weight gain was associated with low LF levels. Hence, food deprivation affected all the measured parameters, and HOC exposure decreased NL levels and lysosomal membrane stability in HG chick liver. Furthermore, autophagic lysosomal parameters have frequently been applied as biomarkers of cellular health status in previous studies of marine and terrestrial invertebrates, and this study suggests that these parameters may be good candidates for biomarkers of cellular health status in seabirds as well.