Intra- and inter-specific variation in metabolic gene expression in relationship to environmental hypoxia

In this study, we explored how environmental oxygen levels affect the metabolic phenotype of sympatric sunfish known to differ in their hypoxia tolerance. We examined bluegill (Lepomis macrochirus) and pumpkinseed (Lepomis gibbosus), two species commonly found in the same water bodies, though pumpkinseed are considered more hypoxia tolerant, and survive in hypoxic lakes that exclude bluegill. Freshly caught Lake Opinicon pumpkinseed possessed significantly higher glycolytic enzyme activities (PGI, ALD, GAPDH, ENO, and LDH) than bluegill, but after holding the fish in an oxygenated environment for 7days, pumpkinseed glycolytic enzymes (PGI, ALD, and LDH) and mRNA (LDHA and HIF1α) declined to bluegill's levels. When glycolytic enzymes and mRNA were compared in pumpkinseed populations from seven lakes, only Penyck Lake pumpkinseed had significantly elevated glycolytic enzyme activity that did not diminish with normoxic holding. The levels of mRNA for LDHA and HIF1α did not differ between lakes and did not change in response to normoxic holding in the Penyck Lake fish. Collectively, these studies on sunfish show that hypoxia tolerance contributes to ecological niche specialization between species, and provides an example of a population that has adapted chronically elevated glycolytic enzyme activity independent of current dissolved oxygen in the water.     

Differential sensitivity of CYP1A to 3,3′,4′,4-tetrachlorobiphenyl and benzo(a)pyrene in two Lepomis species

Although Lepomis species are abundant in a wide variety of habitats throughout North America and could serve as potentially valuable biomonitoring tools, few studies have examined the induction of pollutant biomarkers in this genus. We hypothesized that the induction of cytochrome P-450 1A (CYP1A), a sensitive and widely used indicator of response to aquatic contaminants, would serve as an effective biomarker of organic pollutant exposure in Lepomis species. We examined the response of CYP1A and two of the major pollutant-responsive phase II enzymes, glutathione S-transferase (GST), and uridine diphosphate glucuronyltransferase (UDPGT), in Lepomis exposed to organic pollutants under laboratory and field conditions. Two Lepomis species (longear sunfish, Lepomis megalottis and bluegill, Lepomis macrochirus) were exposed in the laboratory via intraperitoneal injection to corn oil (vehicle), benzo(a)pyrene (BaP) (10 and 50 mg/kg), a polynuclear aromatic hydrocarbon (PAH) or 3,4,3′,4′-tetrachlorobiphenyl (PCB 77) (0.1 and 1.0 mg/kg), a dioxin-like planar halogenated aromatic hydrocarbon (HAH), and sacrificed 2 (BaP) or 7 (corn oil, PCB77) days later. Lepomis hepatic CYP1A exhibited differential sensitivity to these two classes of environmental contaminants. CYP1A activity was weakly induced in bluegill exposed to 1.0 mg/kg PCB 77 (3 fold induction over controls) but strongly induced in both bluegill and longear sunfish exposed to 50 mg/kg BaP (37 and 15 fold induction over controls, respectively). In contrast, hepatic GST activity in both species remained unchanged following the treatment with either compound and hepatic UDPGT activity, which was assessed only in BaP-treated longear sunfish, was unaffected by that chemical, indicating these phase II enzymes may not be sensitive pollutant biomarkers in this genus. Further, longear sunfish collected from a PCB contaminated site displayed relatively low levels of CYP1A activity despite PCB body burdens associated with strong induction of CYP1A activity in other fish species. The strong induction of CYP1A by BaP with much weaker CYP1A response to PCB indicates that CYP1A in Lepomis sp. could be an excellent biomarker for PAH pollution, but may not be a reliable indicator of site contamination by halogenated hydrocarbons. We conclude that Lepomis species provide a useful model for examining the regulation and potential consequences of differential pollutant sensitivity, but that CYP1A in these species should be used with caution as an indicator of halogenated contaminants.     

Stress and interpopulation variation in glycolytic enzyme activity and expression in a teleost fish Fundulus heteroclitus

Northern populations of Fundulus heteroclitus have twofold greater activity of lactate dehydrogenase-B (LDH-B) than southern populations, but exposure to stress increases LDH-B in southern populations, abolishing this difference. To test whether differences in the activity of other hepatic glycolytic enzymes between populations are sensitive to stress, we injected fish with a pharmacological dose of cortisol in coconut oil (400 microg g(-1)) or exposed them to handling stress and measured the activities of all the glycolytic enzymes. At rest, liver phosphofructokinase (PFK) and aldolase (ALD) activities were greater in southern fish, whereas LDH-B activity was greater in northern fish. No other glycolytic enzymes differed in activity between populations in control fish. Cortisol injection and handling stress decreased PFK and ALD and increased LDH activities in the southern but not the northern population, such that the populations no longer differed in the activity of any enzyme following treatment. Unlike Ldh-B mRNA, Pfk and Ald mRNA levels did not parallel enzyme activity, suggesting complex kinetics or regulation at multiple levels. Plasma cortisol did not differ between populations at rest but was significantly different between populations in treated fish. These data suggest that differences in liver enzyme activity may be related to differences in stress hormone physiology between populations.     

Hypoxia regulates avian cardiac Arnt and HIF-1alpha mRNA expression

The aryl hydrocarbon receptor nuclear translocator (Arnt) and hypoxia-inducible factor (HIF)-1alpha mediate cellular responses to hypoxia. We investigated the ability of hypoxia to regulate Arnt and HIF-1alpha mRNA in the heart in vivo. We cloned avian Arnt, developed an in vivo model of chronic cardiac hypoxia, and measured expression of cardiac Arnt and HIF-1alpha mRNA by quantitative RT-PCR. Chronic hypoxic exposure (24 h to 15% O(2)) of day 9 chick embryos resulted in a 30-fold increase in covalent binding of (3)H-misonidazole, a hypoxic tissue marker, to cardiac tissue, and a 2-fold induction of cardiac inducible nitric oxide synthase mRNA, compared to normoxic controls. In this same model, cardiac Arnt mRNA expression decreased by 35%, while HIF-1alpha mRNA expression increased 400%. These data suggest that regulation of Arnt and HIF-1alpha mRNA expression may contribute to the physiological responses of the heart during prolonged hypoxia.     

Temporal dynamics in growth and white skeletal muscle composition of the mummichog Fundulus heteroclitus during chronic hypoxia and hyperoxia

Specific growth rate (G(S) ) and white skeletal muscle composition were measured in the mummichog Fundulus heteroclitus over a period of 28 days at four levels of dissolved oxygen (DO): severe hypoxia (c. 1.2 mg O(2) l(-1) ), moderate hypoxia (3.0 mg O(2) l(-1) ), normoxia (7.1 mg O(2) l(-1) ) and hyperoxia (10.6 mg O(2) l(-1) ). The G(S) was calculated over 0-8, 0-14, 0-28 and 14-28 days, and muscle protein, lactate dehydrogenase (LDH), DNA, RNA and water were measured at 0, 8, 14 and 28 days. Exposure of fish to severe hypoxia was associated with significantly reduced G(S) , lower muscle protein content and lower RNA:DNA compared with other DO treatments. When calculated over the first and second half of the 28 day exposure, however, G(S) of fish in severe hypoxia increased significantly during the second two-week interval, to the same rate as that of normoxic fish. Muscle LDH activity and water content were not significantly affected by DO level. Neither moderate hypoxia nor hyperoxia significantly affected G(S) or any biochemical variable. The results demonstrate that F. heteroclitus can tolerate wide variation in ambient oxygen concentration and, during prolonged exposure to severe hypoxia, shows significant compensation for the initial negative effects on growth. The capacity of F. heteroclitus to grow over a wide range of DO probably contributes to its ability to exploit habitats characterized by marked variation in oxygen availability.     

Effects of hypoxia exposure on hepatic cytochrome P450 1A (CYP1A) expression in Atlantic croaker: molecular mechanisms of CYP1A down-regulation

Hypoxia-inducible factor-α (HIF-α) and cytochrome P450 1A (CYP1A) are biomarkers of environmental exposure to hypoxia and organic xenobiotic chemicals that act through the aryl hydrocarbon receptor, respectively. Many aquatic environments heavily contaminated with organic chemicals, such as harbors, are also hypoxic. Recently, we and other scientists reported HIF-α genes are upregulated by hypoxia exposure in aquatic organisms, but the molecular mechanisms of hypoxia regulation of CYP1A expression have not been investigated in teleost fishes. As a first step in understanding the molecular mechanisms of hypoxia modulation of CYP1A expression in fish, we characterized CYP1A cDNA from croaker liver. Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 2 to 4 weeks) caused significant decreases in hepatic CYP1A mRNA and protein levels compared to CYP1A levels in fish held in normoxic conditions. In vivo studies showed that the nitric oxide (NO)-donor, S-nitroso-N-acetyl-DL-penicillamine, significantly decreased CYP1A expression in croaker livers, whereas the competitive inhibitor of NO synthase (NOS), N(ω)-nitro-L-arginine methyl ester, restored CYP1A mRNA and protein levels in hypoxia-exposed (1.7 mg DO/L for 4 weeks) fish. In vivo hypoxia exposure also markedly increased interleukin-1β (IL-1β, a cytokine), HIF-2α mRNA and endothelial NOS (eNOS) protein levels in croaker livers. Pharmacological treatment with vitamin E, an antioxidant, lowered the IL-1β, HIF-2α mRNA and eNOS protein levels in hypoxia-exposed fish and completely reversed the down-regulation of hepatic CYP1A mRNA and protein levels in response to hypoxia exposure. These results suggest that hypoxia-induced down-regulation of CYP1A is due to alterations of NO and oxidant status, and cellular IL-1β and HIF-α levels. Moreover, the present study provides the first evidence of a role for antioxidants in hepatic eNOS and IL-1β regulation in aquatic vertebrates during hypoxic stress.     

Interactive effects of development and hypoxia on catecholamine synthesis and cardiac function in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>)

The rate-limiting enzyme in the biosynthetic pathway of catecholamines is tyrosine hydroxylase (TH), the activity of which is dependent on molecular oxygen. Zebrafish (Danio rerio) possess two non-allelic TH coding genes, TH1 and TH2. A principal goal of the present study was to determine if the expression of these genes is sensitive to environmental hypoxia. Additionally, we sought to determine if catecholamine content of larvae was changed by environmental hypoxia, and whether the hearts of hypoxic larvae were equally responsive to exogenous catecholamine (norepinephrine) exposure. After 2 days of exposure to hypoxia [5–7 days post-fertilization (dpf); PO₂ = 30 Torr] TH2 mRNA expression was significantly lower and dopamine β hydroxylase (DβH) mRNA was significantly higher in whole larvae. Whole body catecholamine levels were unchanged until after 4 days of hypoxic exposure (5–9 dpf), at which time there was a significant increase in epinephrine and norepinephrine contents. Norepinephrine content was significantly elevated in the hearts of adult fish after 2 and 4 days of hypoxic exposure, and TH1 mRNA expression was increased in the kidney of both groups. After 2 or 4 days of exposure to hypoxia, larvae displayed significantly lower heart rates than normoxic fish. However, application of exogenous norepinephrine caused similar increases in heart rate in both groups. Overall, it is concluded that the mRNA expression of TH1 and TH2 is differentially affected by hypoxia exposure in larvae and adults. Also, catecholamine biosynthesis appears to be activated by 2 dpf and although whole body catecholamine levels increase during hypoxia (possibly promoting downregulation of cardiac β-adrenergic receptors), there is no accompanying decrease in the response of the heart to adrenergic stimulation.     

Tumour necrosis factor-α suppresses the hypoxic response by NF-κB-dependent induction of inhibitory PAS domain protein in PC12 cells

Inflammation is often accompanied by hypoxia. However, crosstalk between signalling pathways activated by inflammation and signalling events that control adaptive response to hypoxia is not fully understood. Here we show that exposure to tumour necrosis factor-α (TNF-α) activates expression of the inhibitory PAS domain protein (IPAS) to suppress the hypoxic response caused by hypoxia-inducible factor (HIF)-1 and HIF-2 in rat pheochromocytoma PC12 cells but not in human hepatoma Hep3B cells. This induction of IPAS was dependent on the nuclear factor-κB (NF-κB) pathway and attenuated hypoxic induction of HIF-1 target genes such as tyrosine hydroxylase (TH) and vascular endothelial growth factor (VEGF). HIF-dependent reporter activity in hypoxia was also decreased following TNF-α treatment. Knockdown of IPAS mRNA by small interfering RNA (siRNA) restored the TNF-α-suppressed hypoxic response. These results indicate that TNF-α is a cell-type specific suppressor of HIFs and suggest a novel crosstalk between stimulation by inflammatory mediators and HIF-dependent hypoxic response.     

Investigation of glucose catabolism in hypoxic Mcf 7 breast cancer culture

Hypoxia plays an important role in tumor phenotype and progression and alters glycolysis, with changes in signaling pathways that develop in response to hypoxia. In this study, the effects of oxygen (normoxia/hypoxia) and of glucose levels on the glucose metabolism was investigated in MCF-7 cancer cells. Under either normoxia or hypoxia conditions, the cells were exposed to glucose at different concentrations (0, 5.5, 15 or 55 mM) for either 3, 6, 12, 24 or 48 h. In all groups, cell viability, levels of key enzymes reflecting glycolytic metabolism in cell lysates, glucose consumed in the medium and extracellular lactate levels and wound closure percentages were determined. In hypoxic cells, intracellular consumption of glucose, and extracellular lactate levels due to increased glucose concentration were observed to be higher (compared to normoxia) and as a result of prolonged exposure to hypoxia, cells were observed to develop resistance to the prolonged exposure to hypoxia. The number of glycolytic enzymes obtained at different levels proved that cells had different potential capacities and changing mechanisms for the metabolic needs of the cell depending on the glucose amount in the medium and time in adapting to the oxygen tension. This study showed that there was an important interaction between hypoxia and glucose metabolism in general, and it was concluded that metabolic processes activated by hypoxia could offer new therapeutic targets.     

Exposure to polychlorinated biphenyls causes endothelial cell dysfunction

Environmental chemicals, such as polychlorinated biphenyls (PCBs), may be atherogenic by disrupting normal functions of the vascular endothelium. To investigate this hypothesis, porcine pulmonary artery-derived endothelial cells were exposed to 3,3′,4,4′-tetrachlorobiphenyl (PCB 77), 2,3,4,4′,5-pentachlorobiphenyl (PCB 114), or 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153) for up to 24 hours. These PCBs were selected for their varying binding avidities with the aryl hydrocarbon (Ah) receptor and differences in their induction of cytochrome P450. PCB 77 and PCB 114 significantly disrupted, in a dose-dependent manner, endothelial barrier function by allowing an increase in albumin transfer across endothelial monolayers. These PCBs also contributed markedly to cellular oxidative stress, as measured by 2,7-dichlorofluorescin (DCF) fluorescence and lipid hydroperoxides, and caused a significant increase in intracellular calcium ([Ca²⁺]_i) levels. Enhanced oxidative stress and [Ca²⁺]_i in PCB 77- and PCB 114-treated cells were accompanied by increased activity and content of cytochrome P450 1A and by a decrease in the vitamin E content in the culture medium. In contrast to the effects of PCB 77 and PCB 114, cell exposure to PCB 153 had no effect on cellular oxidation, [Ca²⁺]_i, or endothelial barrier function. These results suggest that certain PCBs may play a role in the development of atherosclerosis by causing endothelial cell dysfunction and a decrease in the barrier function of the vascular endothelium. It is possible that interaction of PCBs with the Ah receptor and activation of the cytochrome P450 1A subfamily are involved in this pathology. © 1995 John Wiley & Sons, Inc.