Proinflammatory effects of copper deficiency on neutrophils and lung endothelial cells

Dietary copper deficiency increases the accumulation of circulating neutrophils in the rat lung microcirculation. This process includes neutrophil adhesion to, migration along, and emigration though the vascular endothelium. The current study was designed to examine the role of copper in each of these steps. Neutrophils were isolated from rats fed either a copper-adequate (CuA, 6.1 microg Cu/g diet) or copper-deficient diet (CuD, 0.3 microg Cu/g diet) for 4 weeks. First, transient and firm adhesion of neutrophils to P-selectin in a flow chamber showed there were more adhered CuD neutrophils than CuA ones. This effect is probably caused by the increased expression of CD11b that was observed in the current study. Second, the evaluation of neutrophil migration under agarose showed that the CuD neutrophils moved farther than the CuA group in response to IL-8 but not fMLP; this suggests an increased sensitivity to a CD11/CD18-independent signalling pathway. Third, the contractile mechanism of endothelial cells was studied. Elevated F-actin formation in Cu-chelated lung microvascular endothelial cells suggests that neutrophil emigration may be promoted by enhanced cytoskeletal reorganization of the endothelium during copper deficiency. Combined, these results support the theory that dietary copper deficiency has proinflammatory effects on both neutrophils and the microvascular endothelium that promote neutrophil-endothelial interactions.     

Consequences of copper deficiency are not differentially influenced by carbohydrate source in young pigs fed a dired skim milk-based diet

Carbohydrates (CHO) such as fructose (FR) or sucrose, but not starch (ST), aggravate the consequences of dietary copper (Cu) deficiency in rats. To evaluate whether this Cu X CHO interaction is pertinent to human health, the pig was used as an animal model. In two studies, 66 weanling pigs were fed dried skim milk (DSM)-based diets for 10 wk with 20% of the total calories provided as either FR, glucose, or ST and containing either deficient (1.0-1.3 micrograms/g diet) or adequate (7.1 micrograms/g) levels of Cu. Plasma and tissue levels of Cu, the activities of plasma ceruloplasmin ferroxidase and erythrocyte Cu, Zn-superoxide dismutase, and hematocrits were lower (p less than 0.05) in animals fed Cu-deficient diets. The relative cardiac mass of all Cu-deficient groups was greater (p less than 0.05) than that of animals fed Cu-adequate diets. These effects were in general unaffected by type of CHO. For comparison, weaned male rats were also fed DSM-based containing diets ST or FR with adequate or deficient Cu for as long as 10 wk. Rats consuming the Cu-deficient diets were characterized by significantly lower hematocrits, decreased tissue Cu levels, and enlarged hearts, regardless of the CHO source. Together, these data demonstrate that DSM-based diets are not suitable for delineation of potential Cu X CHO interactions, and one or more components of DSM may exacerbate the consequences of dietary Cu deficiency.     

Hearts in adult offspring of copper-deficient dams exhibit decreased cytochrome c oxidase activity, increased mitochondrial hydrogen peroxide generation and enhanced formation of intracellular residual bodies

The long-term effects of low dietary copper (Cu) intake during pregnancy and lactation on cardiac mitochondria in first-generation adult rats was examined. Rat dams were fed diets containing either low (1 mg/kg Cu) or adequate (6 mg/kg Cu) levels of dietary Cu beginning 3 weeks before conception and ending 3 weeks after birth. Cytochrome c oxidase (CCO) activity was 51% lower in isolated cardiac mitochondria from 21-day-old offspring of Cu-deficient dams than in the offspring of Cu-adequate dams. CCO activities in the cardiac mitochondria of 63- and 290-day-old offspring were 22% lower and 14% lower, respectively, in the offspring of Cu-deficient dams after they had been repleted with adequate dietary Cu from the time they were 21 days old. Electron micrographs showed that the size of residual bodies and the cellular volume they occupied in cardiomyocytes rose significantly between 63 and 290 days in the Cu-repleted offspring of Cu-deficient dams, but not in the offspring of Cu-adequate dams. The rate of hydrogen peroxide generation by cardiac mitochondria also was 24% higher in the 290-day-old repleted offspring of Cu-deficient dams than in the offspring of Cu-adequate dams. The increase in hydrogen peroxide production by cardiac mitochondria and in the relative volume and size of dense deposits in cardiomyocytes is consistent with increased oxidative stress and damage resulting from prolonged reduction of CCO activity in the offspring of Cu-deficient dams.     

Relationship between dietary copper concentration and acetylcholine-induced vasodilation in the microcirculation of rats

Dietary copper deficiency has been shown to significantly reduce acetylcholine (Ach)-induced vascular smooth muscle relaxation. The current study was designed to examine the relative relationship between dietary copper and the vasodilator response to Ach in the microcirculation of the rat. Male weanling rats were fed a purified basal diet supplemented with 6.0, 3.0, 1.5 or 0.0 microg Cu/g diet for 4 weeks to provide an adequate, two marginal, and deficient intakes of dietary copper. Arteriole dilation in response to increasing concentrations of acetylcholine (10(-7) to 10(-4) M) was measured in the in vivo cremaster muscle microcirculation for each dietary group. Liver copper and both aortic and erythrocyte Cu,Zn-SOD activity were used as indices of systemic copper status. Dilation to the increasing concentrations of Ach was only different in the 0 microg Cu supplemented group compared to the copper-adequate control values. However, the combined results showed an exponential increase in 10(-5) M Ach-induced vasodilation as liver copper concentration increases from 0 microg Cu/g dry wt. This relationship suggests that dilation is attenuated at liver Cu concentrations below 5 microg/g dry wt. The results indicate that Ach-induced vasodilation is copper-dependent but that the pathway is not very sensitive to short-term marginal restriction of copper intake.     

Neointima formation in the rat carotid artery is exacerbated by dietary copper deficiency

Dietary copper is an essential trace element with roles in both functional and structural aspects of the cardiovascular system. In particular, the vascular response to inflammatory stimuli is known to be significantly augmented in copper-deficient rats. The current study was designed to quantify the extent of injury-induced neointimal proliferation and stenosis in rats fed diets either adequate or deficient in copper. Male, weanling Sprague-Dawley rats were fed purified diets that were either adequate (CuA; 5.6 microg Cu/g) or deficient (CuD; 0.3 microg Cu/g) in copper for 4 weeks. Balloon injury was induced in the left external carotid arteries. Fourteen days after injury, histomorphometric analysis of cross-sections from carotid arteries showed increased neointimal formation in the CuD group compared with the CuA controls (neointima/media ratio: 4.55 +/- 0.93 vs 1.45 +/- 0.2, respectively). These results correspond with data indicating that the activity of Cu/Zn-superoxide dismutase (SOD) is depressed in rats fed this CuD diet. Because superoxide anion and redox status are known to play a key role in the extent of neointimal formation in response to injury, we propose that the exaggerated neointimal proliferation seen in the CuD group is the result of the diminished Cu/Zn-SOD activity.     

Dietary copper deficiency and endothelium-dependent relaxation of rat aorta.

Endothelium-dependent relaxation of aortas was studied in dietary copper (Cu) deficiency. Male, weanling Sprague-Dawley rats were fed diets deficient (CuD, less than 0.5 ppm) or adequate (CuA, 5.0-5.5 ppm) in Cu for 4 weeks. Aortic rings from paired Cu-deficient and Cu-adequate rats were isolated from the descending thoracic aorta, placed in tandem tissue baths, and attached to force transducers. Aortas were contracted with phenylephrine (3 x 10(-7) M) and the degree of force reduction was measured after successively increasing the dose of acetylcholine (10(-8)-10(-5) M), histamine 10(-6)-10(-3) M), or sodium nitroprusside (10(-9)-10(-6) M). Cu deficiency was found to significantly reduce the relaxation responses of each relaxing agent at the highest three of the four doses tested. The ability of Cu-adequate and Cu-deficient aortas to relax was not different, as indicated by their complete relaxation in response to 10(-4) or 10(-5) M papaverine. Because the relaxation responses to both acetylcholine and histamine in rat aorta are dependent on the presence of endothelium, the reduction of these responses suggests that endothelium, or its interaction with smooth muscle, was disrupted in dietary Cu deficiency. The reduction in response to sodium nitroprusside, an endothelium-independent analog of endothelium-derived relaxing factor, indicates that the interaction of endothelium-derived relaxing factor with smooth muscle was disrupted. These findings have implications regarding blood pressure regulation in Cu deficiency.     

Effects of CXC chemokines on neutrophil activation and sequestration in hepatic vasculature

The initiating step of neutrophil-induced cytotoxicity in the liver is the recruitment of these phagocytes into sinusoids. The aim of our study was to compare the efficacy of systemic exposure with individual inflammatory mediators on neutrophil activation and sequestration in the hepatic vasculature of C3Heb/FeJ mice as assessed by flow cytometry and histochemistry, respectively. The CXC chemokine macrophage inflammatory protein-2 (MIP-2; 20 microg/kg) induced a time-dependent upregulation of Mac-1 (318% at 4 h) and shedding of L-selectin (41% at 4 h). MIP-2 treatment caused a temporary increase of sinusoidal neutrophil accumulation at 0.5 h [97 +/- 6 polymorphonuclear leukocytes (PMN)/50 high-power fields (HPF)], which declined to baseline (8 +/- 2) at 4 h. The CXC chemokine KC was largely ineffective in activating neutrophils or recruiting them into the liver. Cytokines (tumor necrosis factor-alpha and interleukin-1alpha) and cobra venom factor substantially increased Mac-1 expression and L-selectin shedding on neutrophils and caused stable sinusoidal neutrophil accumulation (170-220 PMN/50 HPF). Only cytokines induced venular neutrophil margination. Thus CXC chemokines in circulation are less effective than cytokines or complement in activation of neutrophils and their recruitment into the hepatic vasculature in vivo.     

Neutrophil retention in model capillaries: deformability, geometry, and hydrodynamic forces

The initial retention of neutrophils within the pulmonary microvascular bed occurs in both physiological and pathological states, yet the factors responsible for this retention are poorly understood. Because the diameter of the neutrophil is approximately 7.03 micron and the mean pulmonary capillary diameter is 5.5 micron, we postulated that geometric constraints imposed by the microvascular bed, the deformability of the neutrophil, and the hydrodynamic characteristics of blood were important determinants of neutrophil retention. We used a filtration system wherein 111In-labeled human neutrophils (111In-N) suspended in a serum-containing buffer were passed through Nuclepore filters of known pore size. Compared with 99mTc-labeled erythrocytes (99mTc-RBC), the passage of 111In-N was delayed and a higher percentage was retained within the filter. Because the neutrophil and RBC are approximately equal in diameter, the deformability of the neutrophil must be less than that of RBC. As the flow rate increased, retention in the filters decreased logarithmically from 72 +/- 5% (flow rate 0.5 ml/min) to 15 +/- 4% (10.0 ml/min). As the number of RBC in the buffer increased, neutrophil retention in 5-micron filters decreased in a linear fashion from 65 +/- 6% at hematocrit of 0 to 33 +/- 2% at hematocrit of 10. The perfusion pressure and shear stress were of critical importance, and there was a logarithmic relationship between retention and perfusion pressure or shear stress (tau), whether the increase in pressure or tau was generated by increasing flow or by increasing the hematocrit of the perfusate. As the pore size of the filter increased, the retention of neutrophils decreased in a logarithmic fashion: from 75 +/- 5% in the 3-micron filter to 4 +/- 1.3% in the 12-micron filter.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo neutrophil sequestration within lungs of humans is determined by in vitro "filterability".

Neutrophils are normally delayed in transit through the lung microcirculation, relative to the passage of erythrocytes. This sequestration contributes to a pulmonary pool of neutrophils that may relate to the relative inability of neutrophils to deform compared with erythrocytes when in transit in the pulmonary capillaries. A micropore membrane was used to model the human pulmonary microcirculation, in which cell deformability was measured as the pressure developed during filtration of the cells through the membrane at a constant flow. We demonstrated a significant correlation between in vitro deformability and in vivo lung sequestration of indium-111-labeled neutrophils in 10 normal subjects (r = 0.69, P less than 0.02). In eight patients with stable chronic obstructive pulmonary disease, this relationship was not significant (r = -0.2, P greater than 0.05). Furthermore, in a subject with microscopic pulmonary telangiectasia known to allow significant passage of 30-microns microspheres, neutrophils passed through the lungs without delay. Moreover, neutrophils from patients studied acutely with an exacerbation of chronic obstructive pulmonary disease were temporarily less deformable (P less than 0.01). These studies confirm that cell deformability is an important determinant of the normal neutrophil sequestration within the lungs. Changes in cell deformability may alter the extent of this sequestration.     

CXCL1/macrophage inflammatory protein-2-induced angiogenesis in vivo is mediated by neutrophil-derived vascular endothelial growth factor-A

The angiogenic activity of CXC-ELR(+) chemokines, including CXCL8/IL-8, CXCL1/macrophage inflammatory protein-2 (MIP-2), and CXCL1/growth-related oncogene-alpha in the Matrigel sponge angiogenesis assay in vivo, is strictly neutrophil dependent, as neutrophil depletion of the animals completely abrogates the angiogenic response. In this study, we demonstrate that mice deficient in the src family kinases, Hck and Fgr (hck(-/-)fgr(-/-)), are unable to develop an angiogenic response to CXCL1/MIP-2, although they respond normally to vascular endothelial growth factor-A (VEGF-A). Histological examination of the CXCL1/MIP-2-containing Matrigel implants isolated from wild-type or hck(-/-)fgr(-/-) mice showed the presence of an extensive neutrophil infiltrate, excluding a defective neutrophil recruitment into the Matrigel sponges. Accordingly, neutrophils from hck(-/-)fgr(-/-) mice normally migrated and released gelatinase B in response to CXCL1/MIP-2 in vitro, similarly to wild-type neutrophils. However, unlike wild-type neutrophils, those from hck(-/-)fgr(-/-) mice were completely unable to release VEGF-A upon stimulation with CXCL1/MIP-2. Furthermore, neutralizing anti-VEGF-A Abs abrogated the angiogenic response to CXCL1/MIP-2 in wild-type mice and CXCL1/MIP-2 induced angiogenesis in the chick embryo chorioallantoic membrane assay, indicating that neutrophil-derived VEGF-A is a major mediator of CXCL1/MIP-2-induced angiogenesis. Finally, in vitro kinase assays confirmed that CXCL1/MIP-2 activates Hck and Fgr in murine neutrophils. Taken together, these data demonstrate that CXCL1/MIP-2 leads to recruitment of neutrophils that, in turn, release biologically active VEGF-A, resulting in angiogenesis in vivo. Our observations delineate a novel mechanism by which CXCL1/MIP-2 induces neutrophil-dependent angiogenesis in vivo.     

Copper deficiency increases fibulin-5 (DANCE/EVEC) but decreases cytochrome C oxidase VIb subunit expression in rat heart

It has been well documented that dietary copper (Cu) deficiency causes a hypertrophic cardiomyopathy in rodent models. However, a possible alteration in gene expression has not been fully examined. The present study was undertaken to determine the effect of Cu deficiency on protein profiles in rat heart tissue with the combination of the isotope-coded affinity tag (ICAT) method and Western blotting analysis. Male Sprague-Dawley rats were fed diets that were either Cu-adequate (6.0 microg Cu/g diet n=6) or Cu-deficient (0.3 microg Cu/g diet n=6) for 5 week. The ICAT analysis suggested that high-salt buffer (HSB) protein profiles from heart tissue of Cu-deficient rats were different from those of Cu-adequate rats; seven major protein species differed by more than a 100% increase or a 50% decrease. With three available antibodies, our Western blotting analysis confirmed that there was an 85% increase in fibulin-5 (also known DANCE/EVEC) and a 71% decrease in cytochrome C oxidase (CCO) VIb subunit, but no change in succinate dehydrogenase complex (also known complex II) IP subunit in Cu-deficient rat heart. Collectively, these data may be useful in deciphering the molecular basis for the impairments of function related to the hypertrophic-cardiomyopathy of Cu-deficient rats.     

Specific binding and uptake of apolipoprotein E-free high density lipoproteins by cultured liver parenchymal cells of copper-deficient rats

The influence of copper deficiency on the binding and uptake of apolipoprotein E-free high density lipoprotein (apo E-free HDL) in cultured rat hepatic parenchymal cells was examined in this study. Male weanling Sprague-Dawley rats were randomly divided into two treatments, a Cu-adequate (7.33 mg Cu/kg diet) or a Cu-deficient (1.04 mg Cu/kg diet) group. After 7 weeks, plasma apo E-free HDL were isolated by a combination of ultracentrifugation, gel filtration, and heparin-Sepharose affinity chromatography. Parenchymal cells were isolated from collagenase perfused liver of Cu-deficient and adequate rats and cultured for 16 hours at 37 degrees C prior to incubation with iodinated apo E-free HDL from the same treatment group. Cells were incubated with 5 microg/ml(125) I-apo E-free HDL for 2, 6, or 12 hours in the presence or absence of 200 microg/ml (40-fold) excess unlabeled apo E-free HDL. Increases in specific binding at 4 degrees C and specific cell-associated uptake at 37 degrees C as a function of time were observed with cells and HDL from Cu-deficient rats. Cells were also incubated for 6 hours with 8 concentrations of (125)I-apo E-free HDL in the presence or absence of excess unlabeled HDL. Although no significant increase in specific binding was detected at 4 degrees C as a function of ligand concentration, the response tended to be higher at 5 to 15 microg HDL/ml for the Cu-deficient treatment. However, at 37 degrees C the specific cell-associated uptake was increased markedly with cells and HDL from Cu-deficient rats. The observed increases in HDL binding and uptake indicate that these processes may be enhanced in Cu-deficient rats. These data are also consistent with recent in vivo results which indicate that plasma clearance and tissue uptake of HDL are increased in Cu-deficient rats.     

Copper deficiency depresses rat aortae superoxide dismutase activity and prostacyclin synthesis

Prostaglandin synthesis shows dependence on lipid hydroperoxides and resultant oxygen derived radical formation. In view of the importance of dietary copper in cytosolic copper dependent superoxide dismutase (Cu/Zn SOD) activity and the role of SOD in oxygen radical formation, the influence of dietary copper on prostacylin (PGI2) synthesis and SOD activity in rat aorta was examined. Copper deficient (0.5 micrograms Cu/g diet) rats showed a significant 47% reduction in PGI2 synthesis rates by aortic ring incubations in comparison to copper adequate (6.0 micrograms Cu/g diet) animals. Aortic SOD activity was reduced by 46% in copper deficiency in comparison to copper adequate animals. Marginal dietary copper (1.6 micrograms Cu/g diet) significantly reduced aortic SOD activity by 32% but was without effect on aortic ring incubation PGI2 synthesis. These results indicate that dietary copper deficiency, and the resultant decrease in SOD activity, depresses aortic PGI2 synthesis.     

Anti-inflammatory effect of pre-elafin in lipopolysaccharide-induced acute lung inflammation

The aim of the present study was to evaluate the anti-inflammatory activity of pre-elafin, an elastase-specific inhibitor, in lipopolysaccharide (LPS)-induced acute lung inflammation. C57BL/6 mice were pre-treated intranasally with recombinant human pre-elafin or vehicle only. One hour later, they were instilled intranasally with LPS (2 microg/mouse). Animals were sacrificed 6 hours after LPS instillation and bronchoalveolar lavage (BAL) was performed with three 1-ml aliquots of saline. LPS induced a lung inflammation characterised by a 100-fold increase in BAL neutrophils compared to control animals (265.8 +/- 54.5 x 10(3) and 2.4 +/- 1.3 x 10(3) neutrophils/ml, respectively). Pre-elafin dose-dependently reduced the neutrophil influx in the lung alveolar spaces by up to 84%. No elastase activity was detectable in all BAL fluids tested. Pre-elafin also reduced significantly LPS-induced gelatinase activity, as shown by zymography, and BAL macrophage inflammatory protein-2 (MIP-2) and KC levels, two potent neutrophil attractants and activators. Moreover, pre-elafin also significantly reduced mRNA levels of the three members of the IL-1 ligand family, namely IL-1alpha, IL-1beta and IL-1 receptor antagonist (IL-1Ra), type II IL-1 receptor, and TNFalpha as assessed in whole lung tissue by RNase protection assay. Thus, pre-elafin may be considered as a potent anti-inflammatory mediator.     

Development of the stable isotope tracer approach for studies of copper turnover in the rat and mouse

The stable isotope tracer approach was explored for long-term investigations of copper turnover in the adult rat and mouse, with inductively coupled plasma mass spectrometry for isotope measurements. The isotopic measurement method permitted precision and accuracy of <1.0%, with an overall sample blank of <0.05 microg copper. Rats were fed a copper-deficient diet and deionized water with (+Cu) or without (-Cu) copper (20 microg/ml). Both groups underwent a single-day replacement of drinking water with 20 microg/ml of (65)Cu. Compared with the baseline isotope ratio ((65)Cu/(63)Cu) of 0.462 +/- 0.002, blood plasma ratios for the +Cu group on days 2, 7, and 14 postdosing were 0.702 +/- 0.021, 0.557 +/- 0.004, and 0.474 +/- 0.001, respectively. The corresponding data for liver were 1.652 +/- 0.018, 0.560 +/- 0.005, and 0.482 +/- 0.001, respectively. For the -Cu group, respective plasma ratios were 1.580 +/- 0.04. 0.917 +/- 0.02, and 0.664 +/- 0.01 for days 2, 7, and 14 postdosing, and the ratios for liver were 0.987 +/- 0.02, 0.876 +/- 0.04, and 0.739 +/- 0.03. Mice previously made copper deficient to varying degrees were given a single-day replacement with the label. When the 24-hour postdosing isotope ratios in the livers of these mice were correlated with the activity of plasma ceruloplasmin, a negative correlation (r = -0.85) was observed. Isotope enrichment in both rats and mice was greater in the copper-deficient animals compared with the controls.     

A quantitative study of the role of F-actin in producing neutrophil shape

Neutrophils change shape from round to polar and sequentially polymerize/depolymerize actin following chemotactic peptide activation in suspension. To study the relationship between changes in F-actin content and shape we altered the kinetics/extent of actin polymerization and depolymerization with tBOC peptide, cytochalasin D (CD), and low-dose FMLP, and determined the effect of these alterations on the temporal sequence of changes in neutrophil shape. F-actin was measured by FACS analysis of NBDphallacidin-stained cells and expressed as relative fluorescent intensity (RFI) compared to control (RFI = 1.00). Shape was determined by scanning electron microscopy. FMLP causes serial polymerization/depolymerization of actin (RFI = 1.00 +/- 0.04, 1.60 +/- 0.21, 1.10 +/- 0.18, and 1.05 +/- 0.14) associated with four distinct shapes (round-smooth, round-ruffled, blebbed, and polar) noted at 0, 30, 90, 300 sec respectively. Since blebbed and polar shapes appear concurrent with depolymerization and following polymerization, we determined whether depolymerization is required for polarization of cells. The kinetics of depolymerization were: (1) accelerated by tBOC addition at 45 sec, and (2) slowed by high concentrations of FMLP (greater than 10-7M) (300 sec RFI = 1.46). Neither change altered the time course of shape change. To determine whether duration of actin polymerization defines shape, polymerization was halted by addition of tBOC at 5, 10, 20, 30 sec after FMLP to block actin polymerization and shape was monitored at 300 sec. TBOC added 5-20 sec after FMLP limited neutrophil shape change to the blebbed form, while tBOC addition 30 sec following FMLP resulted in a polar shape at 300 sec. To determine whether the extent of actin polymerization affects the shape change sequence, polymerization was limited by (1) inhibition of polymerization with CD, (2) exposure of cells to low concentrations of FMLP (less than 10-9 M), and (3) interruption of polymerization with tBOC. Actin polymerization to RFI less than 1.35-fold basal results in blebbed shape; polymerization greater than 1.35-fold basal yields polar shape. The data show: (1) the human neutrophil demonstrates intermediate shapes when activated by chemotactic peptide, (2) depolymerization of F-actin does not determine shape, and (3) blebbed shape appears when actin polymerizes for greater than 5 sec; polar shape with polymerization greater than or equal to 30 sec to RFI greater than 1.35-fold basal. The data suggest actin polymerization is required for, and extent of polymerization determines, the shape of human neutrophils.     

Copper-deficient rat embryos are characterized by low superoxide dismutase activity and elevated superoxide anions

The teratogenicity of copper (Cu) deficiency may result from increased oxidative stress and oxidative damage. Dams were fed either control (8.0 microg Cu/g) or Cu-deficient (0.5 microg Cu/g) diets. Embryos were collected on Gestational Day 12 for in vivo studies or on Gestational Day 10 and cultured for 48 h in Cu-deficient or Cu-adequate media for in vitro studies. Superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione reductase (GR) activities were measured in control and Cu-deficient embryos as markers of the oxidant defense system. Superoxide anions were measured as an index of exposure to reactive oxygen species (ROS). No differences were found in GPX or GR activities among treatment groups. However, SOD activity was lower and superoxide anion concentrations higher in Cu-deficient embryos cultured in Cu-deficient serum compared to control embryos cultured in control serum. Even so, Cu-deficient embryos had similar CuZnSOD protein levels as controls. In the in vitro system, Cu-deficient embryos had a higher frequency of malformations and increased staining for superoxide anions in the forebrain, heart, forelimb, and somites compared to controls. When assessed for lipid and DNA oxidative damage, conjugated diene concentrations were similar among the groups, but a tendency was observed for Cu-deficient embryos to have higher 8-hydroxy-2'-deoxyguanosine concentrations than controls. Thus, Cu deficiency resulted in embryos with malformations and reduced SOD enzyme activity. Increased ROS concentrations in the Cu-deficient embryo may cause oxidative damage and contribute to the occurrence of developmental defects.     

Copper deficiency and hyperlipoproteinemia induced by a tetramine cupruretic agent in rabbits

The effectiveness of a cupruretic agent, N,N'-bis-(2 amino ethyl)-1,3-propanediamine HCl or 2,3,2-tetramine HCl (TETA), in the induction of copper (Cu) deficiency and the ability of a Cu-deficient diet in the maintenance of the depressed Cu status 10 wk after TETA treatment were examined in this study. In the first experiment, 42 male New Zealand White rabbits, 35 d of age, were randomly divided into three dietary treatments: a copper (Cu)-deficient (2.3 mg Cu/kg diet), a Cu-adequate (13.5 mg Cu/kg diet), and a commercial ration (21.6 mg Cu/kg diet) group. A single oral dose of 100 mg of 2,3,2-tetramine HCl TETA/kg body wt/d were administered to half of the rabbits from each treatment group for 10 d while the remaining rabbits were untreated. In the second experiment, 10 similar rabbits were assigned to three treatments: Cu-deficient plus TETA (n = 4); Cu-adequate plus TETA (n = 3); and Cu-adequate alone (n = 3). The rabbits were fed a TETA dose of 100 mg/d for three 4-d periods over 3 wk, and thereafter maintained on the diets for another 10 wk. Rabbits from the first experiment fed Cu-deficient diet and treated with TETA demonstrated cardiac hypertrophy and markedly reduced plasma and liver Cu concentrations that indicated that the animals were Cu-deficient. Significant elevations (twofold) in low density lipoprotein (LDL) protein, cholesterol, triglyceride, and apolipoprotein B (apo B) concentrations were observed in TETA treated rabbits fed Cu-deficient diet. In the second experiment, the plasma LDL protein level remained elevated, the plasma Cu level was reduced 45%, and the Cu level of the heart when expressed as microgram/g dry tissue was reduced, 10 wk post TETA treatment in rabbits maintained on Cu-deficient diet. Thus, Cu deficiency and hyperlipoproteinemia was rapidly induced by TETA and was still evident 10 wk posttreatment in rabbits maintained on a Cu-deficient diet.     

Interactions of lung stretch, hyperoxia, and MIP-2 production in ventilator-induced lung injury.

The use of positive pressure mechanical ventilation can cause ventilator-induced lung injury (VILI). We hypothesized that hyperoxia in combination with large tidal volumes (VT) would accentuate noncardiogenic edema and neutrophil infiltration in VILI and be dependent on stretch-induced macrophage inflammatory protein-2 (MIP-2) production. In rats ventilated with VT 20 ml/kg, there was pulmonary edema formation that was significantly increased by hyperoxia. Total lung neutrophil infiltration and MIP-2 in bronchoalveolar lavage (BAL) fluid were significantly elevated, in animals exposed to high VT both on room air (RA) and with hyperoxia. Hyperoxia markedly augmented the migration of neutrophils into the alveoli. Anti-MIP-2 antibody blocked migration of neutrophils into the alveoli in RA by 51% and with hyperoxia by 65%. We concluded that neutrophil migration into the alveoli was dependent on stretch-induced MIP-2 production. Hyperoxia significantly increased edema formation and neutrophil migration into the alveoli with VT 20 ml/kg, although BAL MIP-2 levels were nearly identical to VT 20 ml/kg with RA, suggesting that other mechanisms may be involved in hyperoxia-augmented neutrophil alveolar content in VILI.