Mechanisms underlying the health benefits of intermittent hypoxia conditioning.

Intermittent hypoxia (IH) is commonly associated with pathological conditions, particularly obstructive sleep apnoea. However, IH is also increasingly used to enhance health and performance and is emerging as a potent non-pharmacological intervention against numerous diseases. Whether IH is detrimental or beneficial for health is largely determined by the intensity, duration, number and frequency of the hypoxic exposures and by the specific responses they engender. Adaptive responses to hypoxia protect from future hypoxic or ischaemic insults, improve cellular resilience and functions, and boost mental and physical performance. The cellular and systemic mechanisms producing these benefits are highly complex, and the failure of different components can shift long-term adaptation to maladaptation and the development of pathologies. Rather than discussing in detail the well-characterized individual responses and adaptations to IH, we here aim to summarize and integrate hypoxia-activated mechanisms into a holistic picture of the body's adaptive responses to hypoxia and specifically IH, and demonstrate how these mechanisms might be mobilized for their health benefits while minimizing the risks of hypoxia exposure.

Aging Cell Culture - Genetic and Metabolic Effects of Passage Number on Zebrafish Z3 Cells.

BACKGROUND/AIMS: Since cell lines are cultured and extensively used in a variety of different research disciplines, we determined the effects of passage numbers on a commonly used embryonic zebrafish cell line (Z3). METHODS: Senescence markers, DNA damage, the redox state, gene expression, and metabolic parameters have been investigated in young (passage 5) up to very old (passage 40 and higher) cells. RESULTS: Besides increasing DNA damage, we also found elevated metabolic capacity and a shift to a more reduced cellular redox state in the cells. Interestingly, several parameters showed a non-linear course regarding the passage number or cell age, so that for example young and mid-aged cells appeared to cluster with very old rather than with old cells. CONCLUSION: This study illustrates the importance of passage number and suggests pre-testing specific parameters to assure the generation of accurate and reproducible data.

Metabolic Plasticity Enables Circadian Adaptation to Acute Hypoxia in Zebrafish Cells.

BACKGROUND/AIMS: Reduced oxygen availability, hypoxia, is frequently encountered by organisms, tissues and cells, in aquatic environments as well as in high altitude or under pathological conditions such as infarct, stroke or cancer. The hypoxic signaling pathway was found to be mutually intertwined with circadian timekeeping in vertebrates and, as reported recently, also in mammals. However, the impact of hypoxia on intracellular metabolic oscillations is still unknown. METHODS: For determination of metabolites we used Multilabel Reader based fluorescence and luminescence assays, circadian levels of Hypoxia Inducible Factor 1 alpha and oxidized peroxiredoxins were semi quantified by Western blotting and ratiometric quantification of cytosolic and mitochondrial H2O2 was achieved with stable transfections of a redox sensitive green fluorescent protein sensor into zebrafish fibroblasts. Circadian oscillations of core clock gene mRNA´s were assessed using realtime qPCR with subsequent cosine wave fit analysis. RESULTS: Here we show that under normoxia primary metabolic activity of cells predominately occurs during day time and that after acute hypoxia of two hours, administrated immediately before each sampling point, steady state concentrations of glycolytic key metabolites such as glucose and lactate reveal to be highly rhythmic, following a circadian pattern with highest levels during the night periods and reflecting the circadian variation of the cellular response to hypoxia. Remarkably, rhythms in glycolysis are transferred to cellular energy states under normoxic conditions, so that ADP/ATP ratios oscillate as well, which is the first evidence for cycling ADP/ATP pools in a metazoan cell line to our knowledge. Furthermore, the hypoxia induced alterations in rhythms of glycolysis lead to the alignment of three major cellular redox systems, namely the circadian oscillations of NAD+/NADH and NADP+/NADPH ratios and of increased nocturnal levels of oxidized peroxiredoxins, resulting in a highly oxidized nocturnal cellular environment. Of note, circadian rhythms of cytosolic H2O2 remain unaltered, while the transcriptional clock is already attenuated, as it is known to occur also under chronic hypoxia. CONCLUSION: We therefor propose that the realignment of metabolic redox oscillations might initiate the observed hypoxia induced attenuation of the transcriptional clock, based on the reduced binding affinity of the CLOCK/BMAL complex to the DNA in an oxidized environment.

Hypoxia-inducible transcription factors in fish: expression, function and interconnection with the circadian clock.

The hypoxia-inducible transcription factors are key regulators for the physiological response to low oxygen availability. In vertebrates, typically three Hif-α isoforms, Hif-1α, Hif-2α and Hif-3α, are expressed, each of which, together with Hif-1ß, may form a functional heterodimer under hypoxic conditions, controlling expression of hundreds of genes. A teleost-specific whole-genome duplication complicates the analysis of isoform-specific functions in fish, but recent studies suggest that the existence of paralogues of a specific isoform opens up the possibility for a subfunctionalization. In contrast to during development inside the uterus, fish eggs are freely accessible and studies analyzing Hif expression in fish embryos during development have revealed that Hif proteins are not only controlling the hypoxic response, but are also crucial for proper development and organ differentiation. Significant advances have been made in our knowledge about tissue-specific functions of Hif proteins, especially with respect to gill or gonadal tissue. The hypoxia signalling pathway is known to be tightly and mutually intertwined with the circadian clock in zebrafish and mammals. Recently, a mechanistic explanation for the hypoxia-induced dampening of the transcriptional clock was detected in zebrafish, including also metabolically induced alterations of cellular redox signalling. In turn, MAP kinase-mediated H2O2 signalling modulates the temporal expression of Hif-1α protein, similar to the redox regulation of the circadian clock itself. Once again, the zebrafish has emerged as an excellent model organism with which to explore these specific functional aspects of basic eukaryotic cell biology.

Therapeutic nuclear magnetic resonance and intermittent hypoxia trigger time dependent on/off effects in circadian clocks and confirm a central role of superoxide in cellular magnetic field effects.

Cellular magnetic field effects are assumed to base on coherent singlet-triplet interconversion of radical pairs that are sensitive to applied radiofrequency (RF) and weak magnetic fields (WEMFs), known as radical pair mechanism (RPM). As a leading model, the RPM explains how quantum effects can influence biochemical and cellular signalling. Consequently, radical pairs generate reactive oxygen species (ROS) that link the RPM to redox processes, such as the response to hypoxia and the circadian clock. Therapeutic nuclear magnetic resonance (tNMR) occupies a unique position in the RPM paradigm because of the used frequencies, which are far below the range of 0.1-100 MHz postulated for the RPM to occur. Nonetheless, tNMR was shown to induce RPM like effects, such as increased extracellular H2O2 levels and altered cellular bioenergetics. In this study we compared the impact of tNMR and intermittent hypoxia on the circadian clock, as well as the role of superoxide in tNMR induced ROS partitioning. We show that both, tNMR and intermittent hypoxia, exert on/off effects on cellular clocks that are dependent on the time of application (day versus night). In addition, our data provide further evidence that superoxide plays a central role in magnetic signal transduction. tNMR used in combination with scavengers, such as Vitamin C, led to strong ROS product redistributions. This discovery might represent the first indication of radical triads in biological systems.

Environmental and behavioral regulation of HIF-mitochondria crosstalk.

Reduced oxygen availability (hypoxia) can lead to cell and organ damage. Therefore, aerobic species depend on efficient mechanisms to counteract detrimental consequences of hypoxia. Hypoxia inducible factors (HIFs) and mitochondria are integral components of the cellular response to hypoxia and coordinate both distinct and highly intertwined adaptations. This leads to reduced dependence on oxygen, improved oxygen supply, maintained energy provision by metabolic remodeling and tapping into alternative pathways and increased resilience to hypoxic injuries. On one hand, many pathologies are associated with hypoxia and hypoxia can drive disease progression, for example in many cancer and neurological diseases. But on the other hand, controlled induction of hypoxia responses via HIFs and mitochondria can elicit profound health benefits and increase resilience. To tackle pathological hypoxia conditions or to apply health-promoting hypoxia exposures efficiently, cellular and systemic responses to hypoxia need to be well understood. Here we first summarize the well-established link between HIFs and mitochondria in orchestrating hypoxia-induced adaptations and then outline major environmental and behavioral modulators of their interaction that remain poorly understood.

Shaping mechanisms of metal specificity in a family of metazoan metallothioneins: evolutionary differentiation of mollusc metallothioneins.

BACKGROUND: The degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu(+) or Cd(2+). They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order to elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion. RESULTS: HpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd(2+), Zn(2+) or Cu(2+) and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd(6)-HpCdMT and Cu(12)-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by evolutionary modulation of non-cysteine amino acid positions. CONCLUSION: The Roman snail HpCdMT and HpCuMT isoforms can thus be regarded as prototypes of isoform families that evolved genuine metal-specificity within pulmonate molluscs. Diversification into these isoforms may have been initiated by gene duplication, followed by speciation and selection towards opposite needs for protecting copper-dominated metabolic pathways from nonessential cadmium. The mechanisms enabling these proteins to be metal-specific could also be relevant for other metalloproteins.

Quantum based effects of therapeutic nuclear magnetic resonance persistently reduce glycolysis.

Electromagnetic fields are known to induce the clock protein cryptochrome to modulate intracellular reactive oxygen species (ROS) via the quantum based radical pair mechanism (RPM) in mammalian cells. Recently, therapeutic Nuclear Magnetic Resonance (tNMR) was shown to alter protein levels of the circadian clock associated Hypoxia Inducible Factor-1α (HIF-1α) in a nonlinear dose response relationship. Using synchronized NIH3T3 cells, we show that tNMR under normoxia and hypoxia persistently modifies cellular metabolism. After normoxic tNMR treatment, glycolysis is reduced, as are lactate production, extracellular acidification rate, the ratio of ADP/ATP and cytosolic ROS, whereas mitochondrial and extracellular ROS, as well as cellular proliferation are increased. Remarkably, these effects are even more pronounced after hypoxic tNMR treatment, driving cellular metabolism to a reduced glycolysis while mitochondrial respiration is kept constant even during reoxygenation. Hence, we propose tNMR as a potential therapeutic tool in ischemia driven diseases like inflammation, infarct, stroke and cancer.

HIF signaling and overall gene expression changes during hypoxia and prolonged exercise differ considerably.

Exercise as well as hypoxia cause an increase in angiogenesis, changes in mitochondrial density and alterations in metabolism, but it is still under debate whether the hypoxia inducible factor (HIF) is active during both situations. In this study gene expression analysis of zebrafish larvae that were raised under normoxic, hypoxic, or training conditions were compared, using microarray analysis, quantitative real-time PCR and protein data. Although HIF expression is posttranslationally regulated, mRNA expression levels of all three isoforms (HIF-1α, HIF-2α, and HIF-3α) differed in each of the experimental groups, but the changes observed in hypoxic animals were much smaller than in trained larvae. Prominent changes were seen for Hif-2α expression, which significantly increased after the first day of exercise and then decreased down to values significantly below control values. HIF-3α mRNA expression in turn increased significantly, and at the end of the training period (9-15 days postfertilization) it was elevated three times. At the protein level a transient increase in HIF-1α was observed in hypoxic larvae, whereas in the exercise group the amount of HIF-1α protein even decreased below the level of control animals. The analyzed transcriptome was more affected in hypoxic zebrafish larvae, and hardly any genes were similarly altered by both treatments. These results clearly showed that HIF proteins played different roles in trained and hypoxic zebrafish larvae and that the exercise-induced transition to a more aerobic phenotype was not achieved by persistent activation of the hypoxic signaling pathway.

Claudin 28b and F-actin are involved in rainbow trout gill pavement cell tight junction remodeling under osmotic stress.

Permeability of rainbow trout gill pavement cells cultured on permeable supports (single seeded inserts) changes upon exposure to freshwater or treatment with cortisol. The molecular components of this change are largely unknown, but tight junctions that regulate the paracellular pathway are prime candidates in this adaptational process. Using differential display polymerase chain reaction we found a set of 17 differentially regulated genes in trout pavement cells that had been exposed to freshwater apically for 24 h. Five genes were related to the cell-cell contact. One of these genes was isolated and identified as encoding claudin 28b, an integral component of the tight junction. Immunohistochemical reactivity to claudin 28b protein was concentrated in a circumferential ring colocalized to the cortical F-actin ring. To study the contribution of this isoform to changes in transepithelial resistance and Phenol Red diffusion under apical hypo-or hyperosmotic exposure we quantified the fluorescence signal of this claudin isoform in immunohistochemical stainings together with the fluorescence of phalloidin-probed F-actin. Upon hypo-osmotic stress claudin 28b fluorescence and epithelial tightness remained stable. Under hyperosmotic stress, the presence of claudin 28b at the junction significantly decreased, and epithelial tightness was severely reduced. Cortical F-actin fluorescence increased upon hypo-osmotic stress, whereas hyperosmotic stress led to a separation of cortical F-actin rings and the number of apical crypt-like pores increased. Addition of cortisol to the basolateral medium attenuated cortical F-actin separation and pore formation during hyperosmotic stress and reduced claudin 28b in junctions except after recovery of cells from exposure to freshwater. Our results showed that short-term salinity stress response in cultured trout gill cells was dependent on a dynamic remodeling of tight junctions, which involves claudin 28b and the supporting F-actin ring.

Therapeutic Nuclear Magnetic Resonance affects the core clock mechanism and associated Hypoxia-inducible factor-1.

The influence of low intensity electromagnetic fields on circadian clocks of cells and tissues has gained increasing scientific interest, either as a therapeutic tool or as a potential environmental hazard. Nuclear Magnetic Resonance (NMR) refers to the property of certain atomic nuclei to absorb the energy of radio waves under a corresponding magnetic field. NMR forms the basis for Magnetic Resonance Imaging, Magnetic Resonance Spectroscopy and, in a low-intensity form, for NMR therapy (tNMR). Since the circadian clock is bi-directionally intertwined with hypoxic signaling in vertebrates and mammals, we hypothesized that low intensity electromagnetic fields, such as tNMR, might not only affect circadian clocks but also Hypoxia-Inducible Factor-1α (HIF-1α). As master regulator of the hypoxic signaling pathway, HIF-1α is known to dampen the circadian amplitude under reduced oxygen availability, while the hypoxic response of cells and organisms, itself, is tightly clock controlled. In a first experiment, we investigated if tNMR is able to act as Zeitgeber for the core clock mechanism of unsynchronized zebrafish and mouse fibroblast cells, using direct light irradiation and treatment with the glucocorticoid Dexamethasone as references. tNMR significantly affected the cell autonomous clocks of unsynchronized mouse fibroblast cells NIH3-T3, but did not act as a Zeitgeber. Similar to light irradiation and in contrast to treatment with Dexamethasone, tNMR did not synchronize expression profiles of murine clock genes. However, irradiation with tNMR as well as light significantly altered mRNA and protein expression levels of Cryptochrome1, Cryptochrome2 and Clock1 for more than 24 h. Changes in mRNA and protein after different treatment durations, namely 6 and 12 h, appeared to be nonlinear. A nonlinear dose-response relationship is known as hallmark of electromagnetic field induced effects on biological systems. The most prominent alterations were detected in murine HIF-1α protein, again in a nonlinear dose-response. In contrast to murine cells, zebrafish fibroblasts did not respond to tNMR at all. Light, a potent Zeitgeber for the peripheral clocks of fish, led to the expected synchronized clock gene oscillations of high amplitude, as did Dexamethasone. Hence, we conclude, mammalian peripheral clocks are more susceptible to tNMR than the direct light entrainable fish fibroblasts. Although light and tNMR did not act as Zeitgebers for the circadian clocks of unsynchronized murine cells, the significant observed effects might indicate downstream cell-physiological ramifications, which are worth future investigation. However, beside the effects tNMR exerts on the core clock mechanism of mammalian cells, the technology might be the first non-pharmacological approach to modify HIF-1α protein in cells and tissues. HIF-1α and the associated circadian clock play key roles in diseases with underlying ischemic background, such as infarct, stroke, and cancer and, also infectious diseases, such as Covid-19. Hence, low intensity magnetic fields such as tNMR might be of significant medical interest.

Chronic reduction in cardiac output induces hypoxic signaling in larval zebrafish even at a time when convective oxygen transport is not required.

In the present study, the zebrafish breakdance mutant (bre) was used to assess the role of blood flow in development because it has been previously shown that bre larvae have a chronically reduced cardiac output as a result of ventricular contraction following only every second atrial contraction in addition to an atrial bradycardia. We confirmed a 50% reduction compared with control fish and further showed that blood flow in the caudal part of the dorsal aorta decreased by 80%. Associated with these reductions in blood flow were indications of developmental retardation in bre mutants, specifically delayed hatching, reduced cell proliferation, and a transiently decreased growth rate. Surprisingly, an increased red blood cell concentration and an earlier appearance of trunk vessels in bre larvae indicated some compensation to convective oxygen transport, although in previous studies it has been shown that zebrafish larvae at this stage obtain oxygen by bulk diffusion. In bre animals immunohistochemical analyses showed a significant increase in hypoxia inducible factor 1 (HIF)-α protein expression, comparable with wild-type larvae that were raised under hypoxic conditions. Accordingly, the expression of some hif downstream genes was affected. Furthermore, Affymetrix microarray analyses revealed a large number of genes that were differently expressed comparing control and bre larvae, and the number even increased with proceeding development. The results showed that a chronic reduction in blood flow generated hypoxic molecular signals despite partial compensation by increased oxygen carrying capacity and transiently slowed the overall development of zebrafish bre larvae.

Structural and bioinformatic analysis of the Roman snail Cd-Metallothionein gene uncovers molecular adaptation towards plasticity in coping with multifarious environmental stress.

Metallothioneins (MTs) are a family of multifunctional proteins involved, among others, in stress response. The Cadmium (Cd)-MT gene of the Roman snail (Helix pomatia), for example, encodes for a protein induced upon cadmium exposure. While our previous studies have demonstrated that the expressed Cd-MT isoform of Roman snails assists detoxification of cadmium, the present work focuses on the potential plasticity of this gene in response to a variety of environmental stressors playing a crucial role in the specific ecological niche of H. pomatia. Our hypothesis is based on a bioinformatic approach involving gene sequencing, structural and in silico analysis of transcription factor binding sites (TFBs), and a comparison of these features with other MT genes. Our results show that the Roman snail's Cd-MT gene not only is the largest known MT gene, but also contains--apart from the regulatory promoter region--several intronic repeat cassettes of putative TFBs suggested to be involved in environmental stress response, immune competence, and regulation of gene expression. Moreover, intronic scaffold/matrix attachment regions (S/MARs) and stress-induced duplex destabilization sites confer a high potential for epigenetic gene regulation. This suggested regulatory plasticity is also supported by physiological data showing that Cd-MT in Roman snails can be induced differentially not only after cadmium exposure, but also in response to nonmetallic environmental stressors. It is concluded that structural analysis combined with bioinformatic screening may constitute valuable tools for predicting the potential for plasticity and niche-specific adaptation of stress-responsive genes in populations living under rapidly changing environmental conditions.

CFTR gene mutations in pancreatitis: Frequency and clinical manifestations in an Austrian patient cohort.

BACKGROUND/AIMS: Mutations in the gene encoding the cystic fibrosis transmembrane regulator (CFTR) are over-represented in patients with chronic pancreatitis: 13-37% of pancreatitis patients are heterozygous for CFTR mutations, compared with the carrier estimate of 3.2% in the central European population. The aim of the current study was to investigate the association between clinical manifestations of pancreatitis and CFTR carrier status. METHODS: A cohort of 133 pancreatitis patients was recruited in a confined geographical region (Tyrol-Western Austria) and analysed for the 30 most common CFTR gene mutations in Europe by multiplex PCR and gene sequencing. Pancreatitis was classified as acute or chronic according to the criteria of the Japan Pancreas Society (JPS) and etiological factors included in the TIGAR-O classification, namely toxic, idiopathic, genetic, autoimmune, recurrent and obstructive causes were assessed. RESULTS: The overall frequency of CFTR mutations in the patient cohort was 11.2%. In patients classified as 'idiopathic definitive chronic pancreatitis' (JPS criteria), the frequency of mutations was 12.7%, whereas patients with 'acute pancreatitis' or 'possible chronic pancreatitis' (JPS criteria) had a frequency of CFTR mutations of 10% and 9.1%, respectively. CONCLUSION: The frequency of CFTR mutations is highest in patients with definitive chronic pancreatitis and may therefore be regarded as a risk factor for the development of CP. However, multiple etiological factors for pancreatitis are present in the majority of patients. Mutation analysis of the CFTR gene therefore appears to be of limited diagnostic and prognostic value in the management of chronic pancreatitis.

Multiplicity of hypoxia-inducible transcription factors and their connection to the circadian clock in the zebrafish.

In zebrafish, as in most vertebrates, three different isoforms of the hypoxia-inducible transcription factor, Hif-1α, Hif-2α, and Hif-3α, have been identified. The expression data of genes encoding these three proteins, as analyzed so far, show distinct expression patterns for all three isoforms during early development, under hypoxic conditions, and during exercise, suggesting differential roles for all three proteins under these different conditions. While isoform-specific functions for Hif-1α and Hif-2α have been identified in recent years, the role of Hif-3α remains somewhat elusive. Several studies mostly using mammalian cells or tissues discussed Hif-3α as a competitive inhibitor of Hif-1α and Hif-2α. In zebrafish, the expression changes for Hif-1α and Hif-3α observed during development and under environmental stress conditions do not support this hypothesis, and recent studies indicate that Hif-3α is also able to directly control transcriptional activity of certain genes. The Hif signaling pathway is tightly connected to cell circuitries such as glucose and lipid metabolism, and only very recently a further linkage to the circadian clock has been described. In this context a detailed analysis of the mRNA concentrations of hif-1α, hif-2α, and hif-3α also revealed a circadian expression pattern for hif-3α mRNA under normoxic conditions in zebrafish larvae. In addition, accumulation of Hif-1α protein during short-term hypoxia was found to depend on the time within the daily light and dark cycle at which hypoxia was encountered, suggesting that the hypoxia signaling pathway may be regulated by the circadian clock. This is supported by the fact that some of the downstream genes of the Hif signaling pathway, namely, erythropoietin and vascular endothelial growth factor, are known to be clock controlled as well. Furthermore, in developing zebrafish, the disruption of the circadian rhythm was shown to result in a diminished hypoxic response with a modified life cycle of erythrocytes and an altered patterning of the vascular bed, leading to even higher mortality rates of chronodisrupted animals. Hif protein, in turn, is known to affect the circadian clock pathway in zebrafish. Previously, we demonstrated that Hif-1α directly binds to defined E-boxes of the period 1 gene, leading to a sustained dampening of its oscillation amplitude. Here we show that Hif-1α also binds to the promoter of the period 2 gene, indicating that multiple connections between the Hif signaling pathway and the circadian clock exist. The redundancy of the coupling between both pathways might be evidence for the coevolution of both circuits after the great oxygenation event about 2.5 billion years ago. Coupling the circadian clock and the hypoxic signaling pathway may have conferred selective advantages by facilitating a coordinated response of cells and organisms to alternating day-night cycles and concomitant variable food availabilities in the face of varying oxygen supply.

Prolonged hypoxia increases survival even in Zebrafish (Danio rerio) showing cardiac arrhythmia.

Tolerance towards hypoxia is highly pronounced in zebrafish. In this study even beneficial effects of hypoxia, specifically enhanced survival of zebrafish larvae, could be demonstrated. This effect was actually more pronounced in breakdance mutants, which phenotypically show cardiac arrhythmia. Breakdance mutants (bre) are characterized by chronically reduced cardiac output. Despite an about 50% heart rate reduction, they become adults, but survival rate significantly drops to 40%. Normoxic bre animals demonstrate increased hypoxia inducible factor 1 a (Hif-1α) expression, which indicates an activated hypoxic signaling pathway. Consequently, cardiovascular acclimation, like cardiac hypertrophy and increased erythrocyte concentration, occurs. Thus, it was hypothesized, that under hypoxic conditions survival might be even more reduced. When bre mutants were exposed to hypoxic conditions, they surprisingly showed higher survival rates than under normoxic conditions and even reached wildtype values. In hypoxic wildtype zebrafish, survival yet exceeded normoxic control values. To specify physiological acclimation, cardiovascular and metabolic parameters were measured before hypoxia started (3 dpf), when the first differences in survival rate occurred (7 dpf) and when survival rate plateaued (15 dpf). Hypoxic animals expectedly demonstrated Hif-1α accumulation and consequently enhanced convective oxygen carrying capacity. Moreover, bre animals showed a significantly enhanced heart rate under hypoxic conditions, which reached normoxic wildtype values. This improvement in convective oxygen transport ensured a sufficient oxygen and nutrient supply and was also reflected in the significantly higher mitochondrial activity. The highly optimized energy metabolism observed in hypoxic zebrafish larvae might be decisive for periods of higher energy demand due to organ development, growth and increased activity. However, hypoxia increased survival only during a short period of development and starting hypoxia before or after this phase reduced survival, particularly in bre animals. Thus, the physiological plasticity, which enables zebrafish larvae to benefit from a hypoxia, occurs only within a narrow developmental window.

Chronodisruption increases cardiovascular risk in zebrafish via reduced clearance of senescent erythrocytes.

The circadian clock and the hypoxic signaling pathway play critical roles in physiological homeostasis as well as in pathogenesis. The bi-directionality of the interaction between both pathways has been shown on physiological and only recently also on molecular level. But the consequences of a disturbed circadian rhythm for the hypoxic response and the cardiovascular system have never been addressed in any organism. Here we show that the hypoxic response of animals subjected to chronodisruption is reduced by approximately 30%, as reflected by decreased expression levels of hypoxia inducible factor 1 and its down-stream target genes erythropoietin, responsible for the generation of red blood cells (RBC) and vascular endothelial growth factor, which is essential for proper vascularization. Beside malformations of their vascular beds, chronodisrupted animals surprisingly revealed elevated numbers of senescent erythrocytes under normoxic conditions, due to a reduced clearance rate via apoptosis. Over-aged erythrocytes in turn are characterized by decreased oxygen transport capacities and an increased tendency for aggregation, explaining the higher mortality of chronodisrupted animals observed in our study. The present study shows for the first time that chronodisruption strongly interferes with the hypoxic signalling cascade, increasing the cardiovascular risk in zebrafish due to elevated proportions of senescent erythrocytes. The results might shed new light on the etiology of the increased cardiovascular risk observed among shiftworkers.

Linking oxygen to time: the bidirectional interaction between the hypoxic signaling pathway and the circadian clock.

The circadian clock and the hypoxic signaling pathway play critical roles in physiological homeostasis as well as in tumorgenesis. Interactions between both pathways have repeatedly been reported for mammals during the last decade, the molecular basis, though, has not been identified so far. Expression levels of oxygen-regulated and circadian clock genes in zebrafish larvae (Danio rerio) and zebrafish cell lines were significantly altered under hypoxic conditions. Thus, long-term hypoxic incubation of larvae resulted in a dampening of the diurnal oscillation amplitude of the period1 gene expression starting only several hours after start of the hypoxic incubation. A significant decrease in the amplitude of the period1 circadian oscillation in response to hypoxia and in response to the hypoxic mimic CoCl2 was also observed using a zebrafish luciferase reporter cell line in constant darkness. In addition, activity measurements of zebrafish larvae using an infrared-sensitive camera demonstrated the loss of their usual circadian activity pattern under hypoxic conditions. To explore the functional basis of the observed cross-talk between both signaling pathways ChIP assays were performed. Increasing with the duration of hypoxia, a nearly 4-fold occupancy of hypoxia-inducible factor 1 (Hif-1α) at two specific E-box binding sites located in the period1 gene control region was shown, demonstrating therewith the transcriptional co-regulation of the core clock gene by the major transcription factor of the hypoxic pathway. On the other hand, circadian transgenic zebrafish cells, simulating a repressed or an overstimulated circadian clock, modified gene transcription levels of oxygen-regulated genes such as erythropoietin and vascular endothelial growth factor 165 and altered the hypoxia-induced increase in Hif-1α protein concentration. In addition, the amount of Hif-1α protein accumulated during the hypoxic response was shown to depend on the time of the day, with one maximum during the light phase and a second one during the dark phase. The direct binding of Hif-1α to the period1 gene control region provides a mechanistic explanation for the repeatedly observed interaction between hypoxia and the circadian clock. The cross-talk between both major signaling pathways was shown for the first time to be bidirectional and may provide the advantage of orchestrating a broad range of genes and metabolic pathways to cope with altered oxygen availabilities.

Cd accumulation and Cd-metallothionein as a biomarker in Cepaea hortensis (Helicidae, Pulmonata) from laboratory exposure and metal-polluted habitats.

Cepaea hortensis is a widespread terrestrial pulmonate, contributing significantly to element fluxes in soil ecosystems. Due to its capacity of accumulating certain trace elements in its tissues, Cepaea hortensis can serve as a biological indicator of metal accumulation in contaminated areas. In response to Cd exposure this species and related helicid pulmonates are also able to synthesize an inducible, Cd-binding metallothionein (MT) isoform specifically serving in binding and detoxification of this metal. As shown by field-collected garden-snails from a metal-contaminated site near a zinc smelting works in Avonmouth (UK) and an unpolluted reference site in Reutte (Tyrol, Austria), Cd and Cd MT concentrations in midgut gland of C. hortensis from these sites increased with rising Cd concentrations in the soil substrate from the same contaminated sites. By combining the results of these field data with laboratory experiments it appears that midgut gland Cd-MT of Cepaea hortensis seems to fulfil the criteria of a successful biomarker in many respects. First, the synthesis of the protein can rapidly be induced by Cd exposure. Second, the level of Cd MT induction in C. hortensis directly reflects the intensity of metal exposure. Third, the induced signal of increased Cd-MT concentration in C. hortensis is persistent over extended periods of time. Fourth, the Cd-MT signal in C. hortensis seems to be very specific for Cd exposure. Regression analyses demonstrate that tissue levels of Cd and Cd MT in C. hortensis depend on Cd concentrations in the substrate which is represented by either soil or plant material on which snails normally feed. In both cases the best fit for this dependence is exhibited by a semi-logarithmic relationship, with substrate (soil or plant feed) concentrations expressed on a logarithmic scale. It is concluded that C. hortensis and other related pulmonates can successfully be used either as biological indicators of Cd accumulation, or as key species in biomonitoring studies focusing on Cd-MT induction as a biomarker for Cd exposure.