Dolphin leukocytes exhibit an attenuated cytokine response and increase heme oxygenase activity upon exposure to lipopolysaccharides.

Cetaceans exhibit physiological adaptations that allowed the transition to aquatic life, including a robust antioxidant defense system that prevents injury from repeated exposure to ischemia/reperfusion events associated with breath-hold diving. The signaling cascades that characterize ischemic inflammation in humans are well characterized. In contrast, cetaceans' molecular and biochemical mechanisms that confer tolerance to inflammatory events are poorly understood. Heme oxygenase (HO) is a cytoprotective protein with anti-inflammatory properties. HO catalyzes the first step in the oxidative degradation of heme. The inducible HO-1 isoform is regulated by various stimuli, including hypoxia, oxidant stress, and inflammatory cytokines. The objective of this study was to compare the response of HO-1 and cytokines to a proinflammatory challenge in leukocytes isolated from humans and bottlenose dolphins (Tursiops truncatus). We measured changes in HO activity, and abundance and expression of interleukin 1 beta (IL-1ß), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and heme oxygenase 1 (HMOX1) in leukocytes treated with lipopolysaccharide (LPS) for 24 and 48 h. HO activity increased (p < 0.05) in dolphin (48 h) but not human cells. TNF-α expression increased in human (24 h, 48 h), but not dolphin cells following LPS stimulation. LPS-induced cytokine expression was lower in dolphin than in human leukocytes, suggesting a blunted cytokine response in bottlenose dolphin leukocytes treated with LPS. Results suggest species-specific regulation of inflammatory cytokines in leukocytes treated with LPS, which may lead to differential responses to a pro-inflammatory challenge between marine and terrestrial mammals.

In silico Characterization of the Heme Oxygenase 1 From Bottlenose Dolphin (Tursiops truncatus): Evidence of Changes in the Active Site and Purifying Selection.

Cetacea is a clade well-adapted to the aquatic lifestyle, with diverse adaptations and physiological responses, as well as a robust antioxidant defense system. Serious injuries caused by boats and fishing nets are common in bottlenose dolphins (Tursiops truncatus); however, these animals do not show signs of serious infections. Evidence suggests an adaptive response to tissue damage and associated infections in cetaceans. Heme oxygenase (HO) is a cytoprotective protein that participates in the anti-inflammatory response. HO catalyzes the first step in the oxidative degradation of the heme group. Various stimuli, including inflammatory mediators, regulate the inducible HO-1 isoform. This study aims to characterize HO-1 of the bottlenose dolphin in silico and compare its structure to the terrestrial mammal protein. Upstream HO-1 sequence of the bottlenose dolphin was obtained from NCBI and Ensemble databases, and the gene structure was determined using bioinformatics tools. Five exons and four introns were identified, and proximal regulatory elements were detected in the upstream region. The presence of 10 α-helices, three 310 helices, the heme group lodged between the proximal and distal helices, and a histidine-25 in the proximal helix serving as a ligand to the heme group were inferred for T. truncatus. Amino acid sequence alignment suggests HO-1 is a conserved protein. The HO-1 "fingerprint" and histidine-25 appear to be fully conserved among all species analyzed. Evidence of positive selection within an α-helix configuration without changes in protein configuration and evidence of purifying selection were found, indicating evolutionary conservation of the coding sequence structure.

Phosphoenolpyruvate carboxykinase cytosolic and mitochondrial isoforms are expressed and active during hypoxia in the white shrimp Litopenaeus vannamei.

Hypoxic zones in marine environments are spreading around the world affecting the survival of many organisms. Marine animals have several strategies to respond to hypoxia, including the regulation of gluconeogenesis. Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of gluconeogenesis. The objective of this work was to study two isoforms of PEPCK, one mitochondrial (PEPKC-M) and one cytosolic (PEPCK-C), from the white shrimp Litopenaeus vannamei and the response to hypoxia. Both PEPCK isoforms are 72 kDa proteins and have 92% identity at the amino acid level. The mitochondrial isoform has a N-terminal signal peptide for mitochondrial import. Gene expression and enzymatic activity in subcellular fractions were detected in gills, hepatopancreas and muscle in normoxic and hypoxic conditions. Expression of PEPCK-C was higher than PEPCK-M in all the tissues and induced in response to hypoxia at 48 h in hepatopancreas, while the enzymatic activity of PEPCK-M was higher than PEPCK-C in gills and hepatopancreas, but not in muscle and also increased in response to hypoxia in hepatopancreas but decreased in gills and muscle. During limiting oxygen conditions, shrimp tissues obtain energy by inducing anaerobic glycolysis, and although gluconeogenesis implies energy investment, due to the need to maintain glucose homeostasis, these gluconeogenic enzymes are active with contrasting behaviors in the cytosol and mitochondrial cell compartments and appear to be up-regulated in hepatopancreas indicating this tissue pivotal role in gluconeogenesis during the response to hypoxia.