Hypoxia In Zebrafish Research Articles

Combined effects of glyphosate and chemical hypoxia in zebrafish: A new toxicological point of view

Glyphosate (Gly), a systemic and non-selective post-emergence herbicide used worldwide, has emerged as a pollutant. However, its toxic effects are debated by regulatory authorities. In addition, in the aquatic environment, often the presence of pollutants is associated with a hypoxia condition that could change their toxicological effects. We used zebrafish embryos to evaluate the toxic effects of Gly and its mechanisms in a hypoxic condition chemically induced by cobalt chloride (CoCl2). We found that Gly induced toxicity in a time and concentration-dependent manner. The toxicity of Gly was determined at 96 h post fertilization as a lethal concentration (LC), and LC10, LC20, and LC50 values were 85.7, 97, and 122.9 mg/L, respectively. When Gly was combined with CoCl2 the toxicological endpoints were lower than values referred to the Gly alone indicating the worse effects of chemical hypoxia on Gly toxicity. Histological observations were performed at 25, 50, 75, and 100 mg/L for Gly both alone and in combination with 10 mM CoCl2. Fisher's exact test showed significant differences in the presence of hepatic and gut inflammation at 75 and 100 mg/L of Gly both alone and in combination with CoCl2. To deeply investigate the effects of hypoxia on Gly toxicity we decided to test the lowest dose of Gly, 50 mg/L, alone or in combination with CoCl2 10 mM on liver glycogen storage and oxidative stress. Again the results obtained indicate the worse effects of chemical hypoxia on Gly toxicity. Thus Gly toxicity could be reconsidered in light of the damage it causes to the liver and intestines and its effect in combination with factors that induce chemical hypoxia.

Sex matters: Gamete-specific contribution of microRNA following parental exposure to hypoxia in zebrafish

Oxygen availability varies among aquatic environments, and oxygen concentration has been demonstrated to drive behavioral, metabolic, and genetic adaptations in numerous aquatic species. MicroRNAs (miRNAs) are epigenetic modulators that act at the interface of the environment and the transcriptome and are known to drive plastic responses following environmental stressors. An area of miRNA that has remained underexplored is the sex specific action of miRNAs following hypoxia exposure and its effects as gene expression regulator in fishes. This study aimed to identify differences in mRNA and miRNA expression in the F1 generation of zebrafish (Danio rerio) at 1 hpf after either F0 parental male or female were exposed to 2 weeks of continuous (45 %) hypoxia. In general, F1 embryos at 1 hpf demonstrated differences in mRNA and miRNAs expression related to the stressor and to the specific sex of the F0 that was exposed to hypoxia. Bioinformatic pathway analysis of predicted miRNA:mRNA relationships indicated responses in known hypoxia signaling and mitochondrial bioenergetic pathways. This research demonstrates the importance of examining the specific male and female contributions to phenotypic variation in subsequent generations and provides evidence that there is both maternal and paternal contribution of miRNA through eggs and sperm.

Endocrine-Disrupting Chemicals Exposure Alter Neuroendocrine Factors, Disrupt Cardiac Functions and Provokes Hypoxia Conditions in Zebrafish Model.

Zebrafish (Danio rerio) is an increasingly popular vertebrate model used for assessing the toxicity of endocrine-disrupting chemicals (EDCs) on living beings. The zebrafish features high genetic homology to mammals, because of its rapid embryonic development, optical transparency of phenotypic screening embryos, high throughput genetic and chemical screening which make them a powerful toxicological model. This systematic review aimed to assess the recent literature on the use of zebrafish model in EDCs toxicity studies. We capture the data on the types of EDCs used, zebrafish life stages associated with the toxicity, and its effects on the alterations in neuroendocrine factors and cardiac hypoxia in zebrafish. A total of 17 articles published between 2010 and 2020 were curated. The information gathered highlighted the association of EDCs with cardiological outcomes and neurobehavioral effects and distorted expression of genes. The genes that were highlighted in the paper include bdnf, ntrk2a, grin2cb, VTG-1, HIF-1α, tnnt2, ntrk1, and pax6b. The effect of EDCs on cardiac hypoxia and neurodevelopmental and behavioral factors of zebrafish were described in all the papers chosen for this review. The involvement of EDCs in altered regulation of gene expression can be studied further to identify the potential EDC compounds on its toxicological and endocrine disruption function at the molecular level.

Aquatic surface respiration improves survival during hypoxia in zebrafish (Danio rerio) lacking hypoxia-inducible factor 1-α.

Hypoxia-inducible factor 1-α (Hif-1α), an important transcription factor regulating cellular responses to reductions in O2, previously was shown to improve hypoxia tolerance in zebrafish (Danio rerio). Here, we examined the contribution of Hif-1α to hypoxic survival, focusing on the benefit of aquatic surface respiration (ASR). Wild-type and Hif-1α knockout lines of adult zebrafish were exposed to two levels (moderate or severe) of intermittent hypoxia. Survival was significantly compromised in Hif-1α knockout zebrafish prevented from accessing the surface during severe (16 mmHg) but not moderate (23 mmHg) hypoxia. When allowed access to the surface in severe hypoxia, survival times did not differ between wild-type and Hif-1α knockouts. Performing ASR mitigated the negative effects of the loss of Hif-1α with the knockouts initiating ASR at a higher PO2 threshold and performing ASR for longer than wild-types. The loss of Hif-1α had little impact on survival in fish between 1 and 5 days post-fertilization, but as the larvae aged, their reliance on Hif-1α increased. Similar to adult fish, ASR compensated for the loss of Hif-1α on survival. Together, these results demonstrate that age, hypoxia severity and, in particular, the ability to perform ASR significantly modulate the impact of Hif-1α on survival in hypoxic zebrafish.

High protein intake promotes the adaptation to chronic hypoxia in zebrafish (Danio rerio)

Chronic hypoxia is a common environmental stress factor in aquaculture. Many researchers have illustrated the physiological and metabolic changes under chronic hypoxia, but there are few studies to evaluate the relationship between dietary nutrients and hypoxic adaptation in fish. In the present study, we fed zebrafish with control diet (C), high-carbohydrate diet (HC), high-fat diet (HF) and high-protein diet (HP) at normal dissolved oxygen (DO = 7.0 ± 0.5 mg/L) or low dissolved oxygen (DO = 2.5 ± 0.5 mg/L) environment, respectively. After a 7-week feeding trial, the growth performance, body composition, tissue biochemical indexes, mRNA expressions of hifα isoforms and nutrient metabolism-related genes, intestinal digestive enzyme activities and microbiota composition were measured. The results indicated that only the HP diet could alleviate the reduction of body weight and feed efficiency induced by chronic hypoxia, but the HF diet feeding worsened the adverse effects of chronic hypoxia in zebrafish. At the C groups, hypoxia increased catabolism of lipid and protein, but at the HP groups, hypoxia enhanced utilization of glycogen and protein. Compared to the C-hypoxia group, the HP feeding could activate transcription of hif-1a and its downstream glycolysis genes in chronic hypoxia. Furthermore, at hypoxic state, the HP feeding could alleviate the reduction of intestinal digestive enzyme activities and microbiota dysbiosis induced by the chronic hypoxia, including increasing bacterial diversity and decreasing Cetobacterium genus abundance. This is the first study indicating that high protein intake could strengthen ability of fish in resisting the negative effect of chronic hypoxia.

Dopaminergic Co-Regulation of Locomotor Development and Motor Neuron Synaptogenesis is Uncoupled by Hypoxia in Zebrafish.

Hypoxic injury to the developing human brain is a complication of premature birth and is associated with long-term impairments of motor function. Disruptions of axon and synaptic connectivity have been linked to developmental hypoxia, but the fundamental mechanisms impacting motor function from altered connectivity are poorly understood. We investigated the effects of hypoxia on locomotor development in zebrafish. We found that developmental hypoxia resulted in decreased spontaneous swimming behavior in larva, and that this motor impairment persisted into adulthood. In evaluation of the diencephalic dopaminergic neurons, which regulate early development of locomotion and constitute an evolutionarily conserved component of the vertebrate dopaminergic system, hypoxia caused a decrease in the number of synapses from the descending dopaminergic diencephalospinal tract (DDT) to spinal cord motor neurons. Moreover, dopamine signaling from the DDT was coupled jointly to motor neuron synaptogenesis and to locomotor development. Together, these results demonstrate the developmental processes regulating early locomotor development and a requirement for dopaminergic projections and motor neuron synaptogenesis. Our findings suggest new insights for understanding the mechanisms leading to motor disability from hypoxic injury of prematurity.

High-carbohydrate diet promotes the adaptation to acute hypoxia in zebrafish.

Oxygen deprivation (hypoxia) is a common challenge in water environment, which causes lack of energy and oxidative damage in organisms. Many studies have indicated a number of physiological and metabolic changes under hypoxia, but the effects of dietary nutrients on hypoxia tolerance have not been well evaluated. In the present 7-week feeding trial, we fed zebrafish with low-protein diet (LP), high-protein diet (HP), low-fat diet (LF), high-fat diet (HF), low-carbohydrate diet (LC), and high-carbohydrate diet (HC), respectively. Afterward, the resistance to acute hypoxia challenge, growth, body composition, activities of metabolic enzymes, and expressions of energy homeostasis-related genes and six hifαs genes were measured. The results indicated that only the HC diet could significantly improve the resistance to hypoxia challenge. Moreover, the HC diet feeding caused higher glycogen deposition in the liver and muscle, and these glycogens were significantly reduced after 6-h acute hypoxia challenge. Meanwhile, the lactate content in the liver and blood was increased in the HC groups. At hypoxia status, the relative mRNA expressions of the genes related to glycolysis, ATP production, insulin signaling pathway, and hif-3a (hif1al) were all significantly increased in the muscle of the HC diet-fed fish. This study revealed that high-carbohydrate diet could improve the resistance to hypoxia by activating glycolysis and hif/insulin signaling pathway in zebrafish, mainly in the muscle, to efficiently supply energy. Therefore, our results highlight the importance of dietary carbohydrate in resisting hypoxia in fish.

Metabolic plasticity in development: Synergistic responses to high temperature and hypoxia in zebrafish, Danio rerio.

This study investigated interactions of temperature and hypoxia on metabolic plasticity and regulation in zebrafish, Danio rerio, in the first week of development. Larval morphometry, oxygen consumption, and metabolic responses to acute changes in temperature and oxygen were measured in larvae reared under four conditions, including control (28°C and partial pressures of oxygen [PO2] of 21kPa), high temperature (31°C), hypoxia (11kPa), and the two stressors combined. Rearing conditions did not result in consistent morphometric changes; substantial metabolic adjustments, however, were evident. While acute temperature increase resulted in elevated oxygen consumption, with a Q10 of 2.2 ± 0.08, early-staged larvae were able to compensate to chronic temperature rise as routine metabolic rates did not differ between 28°C and 31°C chronic treatments. In contrast, larval responses to chronic and acute hypoxia were similar, with ∼30% decrease in metabolic rates from normoxic values at both temperatures. Further, prior exposure to chronic hypoxia in conjunction with acute high temperature increased Q10 by a factor of 2.5 from 2.2 ± 0.08 to 5.6 ± 0.19. Metabolic suppression by acute hypoxia was independent of any prior exposure conditions. In short, results from this study showed that zebrafish larvae exhibited surprising temperature resilience and metabolic plasticity to a 3°C temperature rise even in their first week of life. Yet exposure to a second stressor (hypoxia) resulted in elevated sensitivity to temperature change that may lead to bioenergetic imbalance due to synergetic effects of temperature and hypoxia on metabolic rates.

Purinergic and Cholinergic Drugs Mediate Hyperventilation in Zebrafish: Evidence from a Novel Chemical Screen.

A rapid test to identify drugs that affect autonomic responses to hypoxia holds therapeutic and ecologic value. The zebrafish (Danio rerio) is a convenient animal model for investigating peripheral O2 chemoreceptors and respiratory reflexes in vertebrates; however, the neurotransmitters and receptors involved in this process are not adequately defined. The goals of the present study were to demonstrate purinergic and cholinergic control of the hyperventilatory response to hypoxia in zebrafish, and to develop a procedure for screening of neurochemicals that affect respiration. Zebrafish larvae were screened in multi-well plates for sensitivity to the cholinergic receptor agonist, nicotine, and antagonist, atropine; and to the purinergic receptor antagonists, suramin and A-317491. Nicotine increased ventilation frequency (fV) maximally at 100 μM (EC50 = 24.5 μM). Hypoxia elevated fV from 93.8 to 145.3 breaths min-1. Atropine reduced the hypoxic response only at 100 μM. Suramin and A-317491 maximally reduced fV at 50 μM (EC50 = 30.4 and 10.8 μM) and abolished the hyperventilatory response to hypoxia. Purinergic P2X3 receptors were identified in neurons and O2-chemosensory neuroepithelial cells of the gills using immunohistochemistry and confocal microscopy. These studies suggest a role for purinergic and nicotinic receptors in O2 sensing in fish and implicate ATP and acetylcholine in excitatory neurotransmission, as in the mammalian carotid body. We demonstrate a rapid approach for screening neuroactive chemicals in zebrafish with implications for respiratory medicine and carotid body disease in humans; as well as for preservation of aquatic ecosystems.

Identification of HIF-1α promoter and expression regulation of HIF-1α gene by LPS and hypoxia in zebrafish

The ubiquitously expressed hypoxia-inducible factor-1α (HIF-1α) acts as a key transcription factor in regulating metabolism, development, cellular survival, proliferation and pathology under hypoxia condition. Compared to mammals, fish are more vulnerable to hypoxia stress and contamination; however, the regulation of HIF-1α in fish remains obscure. In this study, zebrafish HIF-1α promoter was cloned and found to possess a CpG island located at -97 to +403, but the canonical TATA-box was absent. Aligning 240-bp HIF-1α proximal promoter region of zebrafish with other vertebrates showed more than 82 % identity with cyprinid fishes. Further luciferase analysis suggested that the minimal core promoter might locate at -134 to +97, and several putative transcription factor binding sites were found in this region by bioinformatic analysis. Moreover, it was shown that the zebrafish HIF-1α mRNA was significantly activated by 10 μg/mL lipopolysaccharide (LPS) under hypoxia condition and peaked at 8 h after treatment, suggesting LPS- and hypoxia-regulated zebrafish HIF-1α transcriptional activity in a synergistic pattern. This synergistic effect was closely related to the living environment of fish, indicating that this mechanism would be more conducive to fish survival.

Leptin: Clue to poor appetite in oxygen-starved fish

Hypoxia is the most widespread deleterious consequence of eutrophication and has become a major cause of fishery decline. One feature of chronic exposure to hypoxia in fish is inhibition of feeding. In this study, we investigated if the gene that encodes the appetite-suppressing hormone leptin is regulated by hypoxia in zebrafish (Danio rerio). Exposure of adult zebrafish to hypoxic conditions (1+/-0.2 mg O(2) L(-1)) for 4 and 10 days significantly increased leptin-a (zlep-a) mRNA levels in the liver. To evaluate the role of hypoxia-inducible factor 1 (HIF-1) in regulating zlep-a expression, zebrafish embryos were exposed to cobalt chloride (CoCl(2), a HIF-1 inducer) and overexpressed with HIF-1alpha mRNA. Both CoCl(2) treatment and HIF-1alpha overexpression markedly increased zlep-a expression in developing embryos, indicating the possible involvement of HIF-1 in zlep-a regulation. In vivo promoter analysis indicated that zlep-a promoter activity is found in the muscle fibers of zebrafish embryos and enhanced by CoCl(2). This is the first report to show that leptin gene expression in fish is regulated by hypoxia possibly via the involvement of HIF-1.

Tissue-specific transcriptional regulation of monocarboxylate transporters (MCTs) during short-term hypoxia in zebrafish (Danio rerio)

Monocarboxylate transporters (MCTs) have been shown to be important in regulating metabolism during hypoxia in mammals. However, the role of MCTs in hypoxic survival in lower vertebrates is currently unclear. The goal of this study was to investigate the coordinated responses of MCTs along with other metabolic genes during hypoxia. Therefore, we subjected zebrafish (Danio rerio) to 1.5mgL(-1) O(2) over 48 and 96h and measured tissue-specific transcriptional changes of MCTs (1, 2 and 4), lactate dehydrogenase A (LDHa), citrate synthase (CS), and other metabolic proteins using real-time RT-PCR. There were no changes in mRNA detected in muscle at 48 and 96h. When data from both time points were pooled in brain, a significant increase was found in MCT4 (+102%) and LDHa (+28%) mRNA suggesting a preference towards glycolysis. In gills, there were increases in LDHa at 48h (+101%) and MCT1 (+24%) mRNA from pooled data suggesting that both anaerobic and aerobic metabolisms are being utilized. The heart showed the greatest changes in transcriptional regulation compared to other tissues. At 48h, increases were found in MCT1 (+117%), MCT4 (+86%), LDHa (+197%) mRNA, and pooled data showed an increase in CS (+18%) mRNA. These results suggest that the influx and efflux of lactate are both employed as strategies in cardiac tissue during hypoxia. This study has shown that fish utilize tissue-specific transcriptional regulation of MCTs along with other metabolic genes during hypoxia.