P crit – generally defined as the P O 2  below which the animal can no longer maintain a stable rate of O 2 consumption ( Ṁ O 2 ), such that Ṁ O 2  becomes dependent upon P O 2  – provides a single number into which a vast amount of experimental effort has been invested. Here, with specific reference to water-breathers, I argue that this focus on the P crit is not useful for six reasons: (1) calculation of P crit usually involves selective data editing; (2) the value of P crit depends greatly on the way it is determined; (3) there is no good theoretical justification for the concept; (4) P crit is not the transition point from aerobic to anaerobic metabolism, and it disguises what is really going on; (5) P crit is not a reliable index of hypoxia tolerance; and (6) P crit carries minimal information content. Preferable alternatives are loss of equilibrium (LOE) tests for hypoxia tolerance, and experimental description of full Ṁ O 2  versus P O 2  profiles accompanied by measurements of ventilation, lactate appearance and metabolic rate by calorimetry. If the goal is to assess the ability of the animal to regulate Ṁ O 2  from this profile in a mathematical fashion, promising, more informative alternatives to P crit are the regulation index and Michaelis–Menten or sigmoidal allosteric analyses.

The fallacy of the Pcrit - are there more useful alternatives?

Exposure to a variety of environmental factors such as temperature, pH, oxygen and salinity may influence the oxidative status in aquatic organisms. The present review article focuses on the modulation of oxidative stress with reference to the generation of reactive oxygen species (ROS) in aquatic animals from different phyla. The focus of the review article is to explore the plausible mechanisms of physiological changes occurring in aquatic animals due to altered salinity in terms of oxidative stress. Apart from the seasonal variations in salinity, global warming and anthropogenic activities have also been found to influence oxidative health status of aquatic organisms. These effects are discussed with an objective to develop precautionary measures to protect the diversity of aquatic species with sustainable conservation. Comparative analyses among different aquatic species suggest that salinity alone or in combination with other abiotic factors are intricately associated with modulation in oxidative stress in a species-specific manner in aquatic animals. Osmoregulation under salinity stress in relation to energy demand and supply are also discussed. The literature survey of >50 years (1960–2020) indicates that oxidative stress status and comparative analysis of redox modulation have evolved from the analysis of various biotic and/or abiotic factors to the study of cellular signalling pathways in these aquatic organisms.

Modulation of physiological oxidative stress and antioxidant status by abiotic factors especially salinity in aquatic organisms

The physiological causes of intraspecific differences in fitness components such as growth rate are currently a source of debate. It has been suggested that differences in energy metabolism may drive variation in growth, but it remains unclear whether covariation between growth rates and energy metabolism is: (i) a result of certain individuals acquiring and consequently allocating more resources to growth, and/or is (ii) determined by variation in the efficiency with which those resources are transformed into growth. Studies of individually housed animals under standardized nutritional conditions can help shed light on this debate. Here we quantify individual variation in metabolic efficiency in terms of the amount of adenosine triphosphate (ATP) generated per molecule of oxygen consumed by liver and muscle mitochondria and examine its effects, both on the rate of protein synthesis within these tissues and on the rate of whole-body growth of individually fed juvenile brown trout (Salmo trutta) receiving either a high or low food ration. As expected, fish on the high ration on average gained more in body mass and protein content than those maintained on the low ration. Yet, growth performance varied more than 10-fold among individuals on the same ration, resulting in some fish on low rations growing faster than others on the high ration. This variation in growth for a given ration was related to individual differences in mitochondrial properties: a high whole-body growth performance was associated with high mitochondrial efficiency of ATP production in the liver. Our results show for the first time, to our knowledge, that among-individual variation in the efficiency with which substrates are converted into ATP can help explain marked variation in growth performance, independent of food intake. This study highlights the existence of inter-individual differences in mitochondrial efficiency and its potential importance in explaining intraspecific variation in whole-animal performance.

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Differences in mitochondrial efficiency explain individual variation in growth performance

Many fish naturally encounter a daily cycle of hypoxia but it is unclear whether this exposure hardens hypoxia-intolerant fish to future hypoxia or leads to accumulated stress and death. Rainbow trout ( Oncorhynchus mykiss) is a putatively hypoxia-sensitive species found in rivers and estuaries that may routinely experience hypoxic events. Trout were exposed to 1 of 4 135h treatments in a swim-tunnel respirometer: 1) air-saturated control (20.7 kPa PO 2 ); 2) diel cycling O 2 (20.7-4.2 kPa over 24h); 3) acute hypoxia (130h at 20.7 kPa PO 2 followed by 5h at 4.2 kPa PO 2 ); 4) the mean oxygen tension (12.4 kPa PO 2 ) experienced by the diel cycled fish. Some responses were similar in diel O 2 cycled and mean PO 2 -treated fish but overall exposure to ecologically-representative diel hypoxia cycles improved hypoxia tolerance. Diel hypoxia-induced protective responses included increased inducible HSP70 concentration and mean corpuscular hemoglobin concentration, as well as reduced plasma cortisol. Acclimation to diel hypoxia allowed metabolic rates to decline during hypoxia, reduced oxygen debt following subsequent exposures, and allowed fish to return to an anabolic phenotype. The data demonstrate that acute diel cycling hypoxia improves hypoxia tolerance in previously intolerant fish through the activation of cellular protective mechanisms and a reduction in metabolic O 2 requirements.

Diel cycling hypoxia enhances hypoxia-tolerance in rainbow trout (Oncorhynchus mykiss): evidence of physiological and metabolic plasticity

As known, radiation therapy (RT) can exacerbate the degree of hypoxia of tumor cells, which induces serious resistance to RT and in turn, is the greatest obstacle to RT. Reoxygenation can restore the hypoxic state of tumor cells, which plays an important role in reshaping tumor microenviroment for achieving optimal therapeutic efficacy. Herein, we report for the first time that microwave (MW)-triggered IL-Quercetin-CuO-SiO2@ZrO2-PEG nanosuperparticles (IQuCS@Zr-PEG NSPs) have been used to achieve an optimal RT therapeutic outcomes by the strategy of upregulating tumor reoxygenation, i.e. hypoxic cells acquire oxygen and return to normal state. Methods: We prepared a promising multifunctional nanosuperparticle to upregulate tumor reoxygenation by utilizing CuO nanoparticle to generate oxygen under MW irradiation in the tumor microenvironment. The IQuCS@Zr-PEG NSPs were obtained by introducing CuO nanoparticles, MW sensitizer of 1-butyl-3-methylimidazolium hexafluorophosphate (IL), radiosensitizer of Quercetin (Qu) and surface modifier of monomethoxy polyethylene glycol sulfhyl (mPEG-SH, 5k Da) into mesoporous sandwich SiO2@ZrO2 nanosuperparticles (SiO2@ZrO2 NSPs). The release oxygen by IQuCS@Zr-PEG NSPs under MW irradiation was investigated by a microcomputer dissolved oxygen-biochemical oxygen demand detector (DO-BOD) test. Finally, we used the 99mTc-HL91 labeled reoxygenation imaging, Cellular immunofluorescence, immunohistochemistry, and TUNEL experiments to verify that this unique MW-responsive reoxygenation enhancer can be used to stimulate reshaping of the tumor microenvironment. Results: Through experiments we found that the IQuCS@Zr-PEG NSPs can persistently release oxygen under the MW irradiation, which upregulates tumor reoxygenation and improve the combined tumor treatment effect of RT and microwave thermal therapy (MWTT). Cellular immunofluorescence and immunohistochemistry experiments demonstrated that the IQuCS@Zr-PEG NSPs can downregulate the expression of hypoxia-inducible factor 1α (HIF-1α) under MW irradiation. The 99mTc-HL91 labeled reoxygenation imaging experiment also showed that the oxygen generated by IQuCS@Zr-PEG NSPs under MW irradiation can significantly increase the reoxygenation capacity of tumor cells, thus reshaping the tumor microenvironment. The high inhibition rate of 98.62% was achieved in the antitumor experiments in vivo. In addition, the IQuCS@Zr-PEG NSPs also had good computed tomography (CT) imaging effects, which can be used to monitor the treatment of tumors in real-time. Conclusions: The proof-of-concept strategy of upregulating tumor reoxygenation is achieved by MW triggered IQuCS@Zr-PEG NSPs, which has exhibited optimal therapeutic outcomes of combination of RT and MWTT tumor. Such unique MW-responsive reoxygenation enhancer may stimulate the research of reshaping tumor microenvironment for enhancing versatile tumor treatment.

Tumor reoxygenation for enhanced combination of radiation therapy and microwave thermal therapy using oxygen generation in situ by CuO nanosuperparticles under microwave irradiation.

We examined whether oxidative damage and antioxidant responses are more likely to occur during hypoxia or re-oxygenation in hypoxia-tolerant fish, and whether there is an influence of the rate of re-oxygenation. An hypoxia/re-oxygenation experiment using wild-caught Cyphocharax abramoides (Rio Negro, Brazil), was designed to answer these questions. Lipid peroxidation (MDA), a measure of oxidative damage, and antioxidant activities (superoxide dismutase (SOD), glutathione peroxidase (GPx), antioxidant capacity against peroxyl radicals (ACAP)), were measured in brain, gill and liver tissues after normoxia, 3-h hypoxia (2.7 kPa), and 3-h hypoxia followed by 1-h or 3-h re-oxygenation, implemented either immediately or slowly (3.0 kPa·h −1 ). Critical oxygen tension of routine oxygen consumption rate (Pcrit) (4.1 kPa) and the P O2 at loss of equilibrium (LOE) (1.7 kPa) were determined to set the experimental hypoxia exposure. The Regulation Index, a measure of oxyregulation with declining P O2 , was 0.32. Oxidative damage occurred during hypoxia: no additional damage was observed during re-oxygenation. Tissues responded differentially. GPx and MDA rose in the brain and gills, and SOD (and likely GPx) in the liver during hypoxia. Antioxidants increased further at LOE. Rate of oxygen increase during re-oxygenation did not affect antioxidant responses. In brain and gills, GPx and MDA decreased or recovered after 1-h re-oxygenation. In liver, SOD remained high and GPx increased. In summary, C. abramoides incurred oxidative damage during hypoxic exposure with no additional damage inflicted during re-oxygenation: the rate of re-oxygenation was inconsequential. Literature data support conclusion of greater damage during hypoxia than during re-oxygenation in hypoxia-tolerant fish.

Does hypoxia or different rates of re-oxygenation after hypoxia induce an oxidative stress response in Cyphocharax abramoides (Kner 1858), a Characid fish of the Rio Negro?

The Amazonian cichlid, Astronotus ocellatus , is highly tolerant to hypoxia, and is known to reduce its metabolic rate by reducing the activity of energetically expensive metabolic processes when oxygen is lacking in their environment. Our objectives were to determine how protein metabolism is regulated in A. ocellatus during hypoxia. Fish were exposed to a stepwise decrease in air saturation (100%, 20%, 10% and 5%) for 2 hours at each level, and sampled throughout the experiment. A flooding dose technique using a stable isotope allowed us to observe an overall decrease in protein synthesis during hypoxia in liver, muscle, gill and heart. We estimate that this decrease in rates of protein synthesis accounts for a 20 to 36% decrease in metabolic rate, which would enable oscars to maintain stable levels of ATP and prolong survival. It was also determined for the first time in fish that a decrease in protein synthesis during hypoxia is likely controlled by signaling molecules (4EBP1 and eIF2-α), and not simply due to a lack of ATP. We could not detect any effects of hypoxia on protein degradation as the levels of NH 4 excretion, indicators of the ubiquitin proteasome pathway, and enzymatic activities of lysosomal and non-lysosomal proteolytic enzymes were maintained throughout the experiment.

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Protein synthesis is lowered by 4EBP1 and eIF2-α signaling while protein degradation may be maintained in fasting, hypoxic Amazonian cichlids Astronotus ocellatus.

Oxygen-dependent distinct expression of hif-1α gene in aerobic and anaerobic tissues of the Amazon Oscar, Astronotus crassipinnis Part B Biochemistry & molecular biology

Waldir D. Heinrichs-Caldas

Papers

Does hypoxia or different rates of re-oxygenation after hypoxia induce an oxidative stress response in Cyphocharax abramoides (Kner 1858), a Characid fish of the Rio Negro?

Protein synthesis is lowered by 4EBP1 and eIF2-α signaling while protein degradation may be maintained in fasting, hypoxic Amazonian cichlids Astronotus ocellatus.

Oxygen-dependent distinct expression of hif-1α gene in aerobic and anaerobic tissues of the Amazon Oscar, Astronotus crassipinnis Part B Biochemistry & molecular biology