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Extremely anoxia-tolerant animals, such as freshwater turtles, survive anoxia and reoxygenation without sustaining tissue damage to their hearts. In contrast, for mammals, the ischemia–reperfusion (IR) injury that leads to tissue damage during a heart attack is initiated by a burst of superoxide (O2·−) production from the mitochondrial respiratory chain upon reperfusion of ischemic tissue. Whether turtles avoid oxidative tissue damage because of an absence of mitochondrial superoxide production upon reoxygenation, or because the turtle heart is particularly protected against this damage, is unclear. Here, we investigated whether there was an increase in mitochondrial O2·− production upon the reoxygenation of anoxic red-eared slider turtle hearts in vivo and in vitro. This was done by measuring the production of H2O2, the dismutation product of O2·−, using the mitochondria-targeted mass-spectrometric probe in vivo MitoB, while in parallel assessing changes in the metabolites driving mitochondrial O2·− production, succinate, ATP and ADP levels during anoxia, and H2O2 consumption and production rates of isolated heart mitochondria. We found that there was no excess production of in vivo H2O2 during 1 h of reoxygenation in turtles after 3 h anoxia at room temperature, suggesting that turtle hearts most likely do not suffer oxidative injury after anoxia because their mitochondria produce no excess O2·− upon reoxygenation. Instead, our data support the conclusion that both the low levels of succinate accumulation and the maintenance of ADP levels in the anoxic turtle heart are key factors in preventing the surge of O2·− production upon reoxygenation.

Supplementary Data from TW-37, a small-molecule inhibitor of Bcl-2, mediates S-phase cell cycle arrest and suppresses head and neck tumor angiogenesis

Members of the Bcl-2 family play a major role in the pathobiology of head and neck cancer. We have shown that Bcl-2 orchestrates a cross talk between tumor cells and endothelial cells that have a direct effect on the progression of head and neck squamous cell carcinoma (HNSCC). Notably, Bcl-2 is significantly up-regulated in the tumor-associated endothelial cells compared with the endothelial cells of normal oral mucosa in patients with HNSCC. Here, we evaluated the effect of TW-37, a small-molecule inhibitor of Bcl-2, on the cell cycle and survival of endothelial cells and HNSCC and on the progression of xenografted tumors. TW-37 has an IC50 of 1.1 μmol/L for primary human endothelial cells and averaged 0.3 μmol/L for head and neck cancer cells (OSCC3, UM-SCC-1, and UM-SCC-74A). The combination of TW-37 and cisplatin showed enhanced cytotoxic effects for endothelial cells and HNSCC in vitro, compared with single drug treatment. Notably, whereas cisplatin led to an expected G2-M cell cycle arrest, TW-37 mediated an S-phase cell cycle arrest in endothelial cells and in HNSCC. In vivo, TW-37 inhibited tumor angiogenesis and induced tumor apoptosis without significant systemic toxicities. Combination of TW-37 and cisplatin enhanced the time to tumor failure (i.e., 4-fold increase in tumor volume), compared with either drug given separately. Collectively, these data reveal that therapeutic inhibition of Bcl-2 function with TW-37 is sufficient to arrest endothelial cells and HNSCC in the S phase of the cell cycle and to inhibit head and neck tumor angiogenesis.[Mol Cancer Ther 2009;8(4):893–903]

Supplementary Figures from TW-37, a small-molecule inhibitor of Bcl-2, mediates S-phase cell cycle arrest and suppresses head and neck tumor angiogenesis

Background Environmental fluctuation during embryonic and fetal development can permanently alter an organism’s morphology, physiology, and behaviour. This phenomenon, known as developmental plasticity, is particularly relevant to reptiles that develop in subterranean nests with variable oxygen tensions. Previous work has shown hypoxia permanently alters the cardiovascular system of snapping turtles and may improve cardiac anoxia tolerance later in life. The mechanisms driving this process are unknown but may involve epigenetic regulation of gene expression via DNA methylation. To test this hypothesis, we assessed in situ cardiac performance during 2 h of acute anoxia in juvenile turtles previously exposed to normoxia (21% oxygen) or hypoxia (10% oxygen) during embryogenesis. Next, we analysed DNA methylation and gene expression patterns in turtles from the same cohorts using whole genome bisulfite sequencing, which represents the first high-resolution investigation of DNA methylation patterns in any reptilian species. Results Genome-wide correlations between CpG and CpG island methylation and gene expression patterns in the snapping turtle were consistent with patterns observed in mammals. As hypothesized, developmental hypoxia increased juvenile turtle cardiac anoxia tolerance and programmed DNA methylation and gene expression patterns. Programmed differences in expression of genes such as SCN5A may account for differences in heart rate, while genes such as TNNT2 and TPM3 may underlie differences in calcium sensitivity and contractility of cardiomyocytes and cardiac inotropy. Finally, we identified putative transcription factor-binding sites in promoters and in differentially methylated CpG islands that suggest a model linking programming of DNA methylation during embryogenesis to differential gene expression and cardiovascular physiology later in life. Binding sites for hypoxia inducible factors (HIF1A, ARNT, and EPAS1) and key transcription factors activated by MAPK and BMP signaling (RREB1 and SMAD4) are implicated. Conclusions Our data strongly suggests that DNA methylation plays a conserved role in the regulation of gene expression in reptiles. We also show that embryonic hypoxia programs DNA methylation and gene expression patterns and that these changes are associated with enhanced cardiac anoxia tolerance later in life. Programming of cardiac anoxia tolerance has major ecological implications for snapping turtles, because these animals regularly exploit anoxic environments throughout their lifespan.

Increases in ambient salinity demand parallel increases in intestinal base secretion for maintenance of osmoregulatory status, which is likely the cause of a transient acidosis following transfer of euryhaline fish from freshwater to seawater. It was predicted that transfer of the marine Gulf toadfish (Opsanus beta) from seawater (35 ppt) to hypersaline (60 ppt) seawater (HSW) would lead to a transient acidosis that would be compensated by increases in branchial acid excretion to offset the acid–base disturbance. Toadfish exposed to HSW showed a significant decrease in blood pH and [HCO3−] but no increase in pCO2, followed by a full recovery after 48–96 h. A similar metabolic acidosis and recovery was found when fish were exposed to 60-ppt HCO3−-free seawater (HEPES-buffered), which may suggest that compensation for intestinal base loss during hypersaline treatment is from gill H+ excretion rather than gill HCO3− uptake. However, we cannot rule out that reduced branchial HCO3− excretion contributed to an increase in net acid excretion. Since colchicine prevents full compensation, translocation of H+ and/or HCO3− transporters between cytosolic compartments and plasma membrane fractions might be involved in compensating for the hypersalinity-induced acidosis. Translocation of transporters rather than de novo synthesis may represent a faster and less energetically demanding response to rapidly fluctuating and high salinities encountered by toadfish in their natural environment.

Aquaporins are the predominant water-transporting proteins in vertebrates, but only a handful of studies have investigated aquaporin function in fish, particularly in mediating water permeability during salinity challenges. Even less is known about aquaporin function in hypoxia (low oxygen), which can profoundly affect gill function. Fish deprived of oxygen typically enlarge gill surface area and shrink the water-to-blood diffusion distance to facilitate oxygen uptake into the bloodstream. However, these alterations to gill morphology can result in unfavourable water and ion fluxes. Thus, there exists an osmorespiratory compromise, whereby fish must try to balance high branchial gas exchange with low ion and water permeability. Furthermore, the gills of seawater and freshwater teleosts have substantially different functions with respect to osmotic and ion fluxes; consequently, hypoxia can have very different effects according to the salinity of the environment. The purpose of this study was to determine what role aquaporins play in water permeability in the hypoxia-tolerant, euryhaline common killifish (Fundulus heteroclitus), in two important osmoregulatory organs – the gills and intestine. Using immunofluorescence, we localized aquaporin-3 protein to the basolateral and apical membranes of ionocytes and enterocytes, respectively. Although hypoxia increased branchial aquaporin-3 mRNA expression in seawater and freshwater, protein abundance did not correlate. Indeed, hypoxia did not alter aquaporin-3 protein abundance in seawater and reduced it in the cell membranes of freshwater gills. Together, these observations suggest killifish aquaporin-3 contributes to reduced diffusive water flux during hypoxia and normoxic recovery in freshwater, and facilitates intestinal permeability in seawater and freshwater.

It is well established that adult vertebrates acclimatizing to hypoxic environments undergo mitochondrial remodeling to enhance oxygen delivery, maintain ATP, and limit oxidative stress. However, many vertebrates also encounter oxygen deprivation during embryonic development. The effects of developmental hypoxia on mitochondrial function are likely to be more profound, because environmental stress during early life can permanently alter cellular physiology and morphology. To this end, we investigated the long-term effects of developmental hypoxia on mitochondrial function in a species that regularly encounters hypoxia during development—the common snapping turtle (Chelydra serpentina). Turtle eggs were incubated in 21% or 10% oxygen from 20% of embryonic development until hatching, and both cohorts were subsequently reared in 21% oxygen for 8 months. Ventricular mitochondria were isolated, and mitochondrial respiration and reactive oxygen species (ROS) production were measured with a microrespirometer. Compared to normoxic controls, juvenile turtles from hypoxic incubations had lower Leak respiration, higher P:O ratios, and reduced rates of ROS production. Interestingly, these same attributes occur in adult vertebrates that acclimatize to hypoxia. We speculate that these adjustments might improve mitochondrial hypoxia tolerance, which would be beneficial for turtles during breath-hold diving and overwintering in anoxic environments.

Figures and Tables

[Graphic][1] Music is a common language across many cultures and is often celebrated with large festivals that can attract thousands of people in just a single day. For festival-goers, the relentless melodies and rhythms can be exhilarating, but for nearby neighbours, it can be an intrusive

[Graphic][1] In the Canadian North Pole, there exists but one freshwater fish species: the Arctic char ( Salvelinus alpinus ). This keystone species is an indicator of ecosystem health, because it is highly sensitive to environmental changes. And, because many northerners depend on char for

As the final electron acceptor in the electron transport chain, oxygen is vital to produce ATP in the process of oxidative phosphorylation. In most vertebrates, the absence of oxygen (anoxia) quickly leads to an imbalance of ATP supply and demand and the activation of cell–death pathways. Remarkably, however, freshwater turtles of the genera Trachemys and Chrysemys, and fishes of the genus Carassius (crucian carp and the closely related goldfish) can survive in anoxic environments for periods lasting several months. While both species inhabit similar environments, they use very different strategies to survive oxygen deprivation. In this review, we focus on metabolic strategies and adaptations that allow these species to avoid, regulate, or mitigate the negative effects of anoxia and reoxygenation on cell function. Specifically, we review strategies to balance ATP supply and demand, the regulation of toxic end-products, mitochondrial remodelling, metabolite accumulation, regulation of reactive oxygen species, and Ca 2+ management. Through the strategies they use, turtles and crucian carp are excellent model species to shed light on metabolic adaptations to anoxia and reveal novel drug targets for the treatment of oxygen-related diseases in humans.

Increases in ambient salinity demand parallel increases in intestinal base secretion for maintenance of osmoregulatory status, which is likely the cause of a transient acidosis following transfer of euryhaline fish from freshwater to seawater. It was predicted that transfer of the marine Gulf toadfish (Opsanus beta) from seawater (35 ppt) to hypersaline (60 ppt) seawater (HSW) would lead to a transient acidosis that would be compensated by increases in branchial acid excretion to offset the acid-base disturbance. Toadfish exposed to HSW showed a significant decrease in blood pH and [HCO