Maintenance of zinc-dependent hepatic functions in rat hepatocytes cultured in medium without added zinc

Hepatocytes were cultured with Waymouth's media containing zinc at concentrations of 1 (the endogenous zinc concentration of basal medium), 16 and 48 mumols Zn/L to examine the effects of extracellular zinc on a variety of zinc-related functions. The zinc concentrations were chosen with the intention of simulating zinc-deficient, adequate and excess extracellular conditions. Basal medium had no effect on cell zinc, metallothionein (MT) or MTmRNA for up to 48 h but reduced delta-aminolevulinic acid dehydratase (delta-ALA-D) activity to 75% of the initial level by 3 h. The addition of zinc at 16 or 48 mumols Zn/L during the initial 3 h of culture did not prevent the decrease in delta-ALA-D activity. Reintroducing zinc at concentrations of 16 or 48 mumols Zn/L to hepatocytes after the initial 3 h of culture in basal medium significantly increased cell zinc, MT and MTmRNA levels and fully restored delta-ALA-D activity by 24 h. Medium zinc had no apparent effect on membrane integrity assessed as leakage of lactate dehydrogenase activity into culture media or de novo protein synthesis as examined by two-dimensional gel electrophoresis of 35S-labeled proteins. Hepatocytes cultured in basal medium resisted losses in cell zinc concentration even when EDTA and bovine serum albumin were present in culture medium. Kinetic experiments using 65Zn suggest hepatocytes maintain zinc concentrations by reducing zinc efflux. The ability of hepatocytes cultured in basal (1 mumol Zn/L) medium to maintain cell zinc content and some zinc-dependent functions underscores the difficulty of producing zinc deficiency in primary hepatocyte culture.     

Effect of vitamin E on linoleic acid-mediated induction of peroxisomal enzymes in cultured porcine endothelial cells

Linoleic acid decreases endothelial barrier function in culture. We hypothesize that the mechanism may involve induction of peroxisomes, with subsequent generation of hydrogen peroxide, and that vitamin E may protect against barrier function loss by preventing the induction of peroxisomal enzymes. To investigate this hypothesis, we exposed cultured endothelial cells to 0 or 90 mumols/L linoleic acid [18:2(n-6)], with or without 25 mumols/L supplemental vitamin E, for 5 d. The induction of peroxisomes by linoleic acid exposure was determined by measuring cellular peroxisomal beta-oxidation and catalase activity. Vitamin E alone had no effect on beta-oxidation or catalase activity, whereas linoleic acid exposure significantly increased both compared with control values. Vitamin E supplementation prevented induction of peroxisomal beta-oxidation and catalase activity by 18:2. In contrast, cell enrichment with vitamin E had no effect on 18:2-induced accumulation of cytoplasmic lipid-like droplets. These results confirm our hypothesis that the protective effects of vitamin E against fatty acid-mediated endothelial cell injury may be due in part to the ability of vitamin E to prevent the induction of peroxisomal beta-oxidation enzymes and thus the formation of excess hydrogen peroxide.     

Zinc status influences zinc transport by porcine brain capillary endothelial cells

Brain capillary endothelial cells (BCEC) were cultured as an in vitro model of the blood-brain barrier (BBB) and manipulated to investigate how the BBB responds to changes in zinc status. BCEC were grown in minimum essential medium (MEM) with 2% fetal bovine serum and 13% platelet-poor horse serum. A moderate zinc deficiency was imposed by growing the cells in medium containing serums that had previously been dialyzed against EDTA to remove endogenous labile zinc. The control treatment was MEM with undialyzed serums (3 micro mol Zn/L); low-Zn was MEM with dialyzed serums (1.5 micro mol Zn/L); Zn-back was MEM with dialyzed serums, plus ZnCl(2) added back (3 micro mol Zn/L); high-Zn was MEM with undialyzed serums, plus ZnCl(2) (50 micro mol Zn/L). Low-Zn treatment increased (P < 0.02) the rate of zinc uptake into BCEC, relative to control and Zn-back; low-Zn treatment also increased (P < 0.05) the rate of zinc transport across the BCEC into the abluminal chamber (analogous to the brain), relative to control and Zn-back. High-Zn decreased (P < 0.02) the rate of zinc transport across BCEC into the brain, while increasing (P < 0.001) the rate of zinc uptake into BCEC, relative to controls. We conclude that BCEC responded to changes in zinc status by altering the rate of zinc transport in a manner consistent with the BBB actively working to sustain brain zinc homeostasis.     

Effects of dietary zinc depletion on seminal volume and zinc loss, serum testosterone concentrations, and sperm morphology in young men.

Identification of the andrological variables most sensitive to zinc depletion would expedite the diagnosis of male reproductive pathology induced by zinc deficiency. Eleven volunteers living on a metabolic ward were fed a diet composed of a mixture of a semisynthetic formula and conventional foods supplemented with ZnSO4 to supply a total of 1.4, 2.5, 3.4, 4.4, or 10.4 mg Zn/d. After an equilibration period of 28 d (10.4 mg Zn/d), all treatments were presented for 35 d each, the first four in random order and the fifth last. Compared with when they were consuming 10.4 mg Zn/d, volunteers consuming 1.4 mg Zn/d exhibited decreased semen volumes (3.30 vs 2.24 mL) and serum testosterone concentrations (26.9 vs 21.9 nmol/L), and no change in seminal zinc concentrations. Compared with 10.4 mg Zn/d, treatments of 1.4, 2.5, and 3.4 mg Zn/d decreased the total semen zinc loss per ejaculate (6.29 vs 3.81, 4.68, and 5.03 mumols/ejaculate). Seminal loss accounted for 9% of total body zinc loss when 1.4 mg Zn/d was consumed. Seminal phosphorus concentrations were elevated during all four phases of zinc depletion (28.4 vs 32.9, 31.0, 34.2, and 33.6 mmol/L). The findings suggest that serum testosterone concentrations, seminal volume, and total seminal zinc loss per ejaculate are sensitive to short-term zinc depletion in young men.     

用小鼠肢芽器官培养技术研究锌缺乏与过量的致畸性

应用小鼠胚胎肢芽器官体外培养和计算机图像分析技术，通过体内／外和体外实验途径研究锌缺乏与过量的致畸性。结果表明：缺锌使多数肢体软骨原基发育受阻，软骨面积减小，外形发育受抑制，出现严重的缺指（趾）和并指（趾）。软骨和软组织均受影响。后肢比前肢受到的影响大；随着锌剂量的过度增加，肢体中各种软骨原基的发育分化越差、软骨面积越小，呈剂量反应关系。后肢比前肢易受影响，爪骨比长骨敏感，骨组织受到的影响比软组织严重。在体外实验中，锌含量在１０～１５ｍｇ／Ｌ（培养基）显示了致畸性。在体内／外实验中，５０ｍｇ／ｋｇｂｗ就开始对肢体产生影响。     

Zinc protects against apoptosis of endothelial cells induced by linoleic acid and tumor necrosis factor alpha

BACKGROUND: Zinc requirements of the vascular endothelium may be increased in inflammatory conditions, ie, atherosclerosis, in which apoptotic cell death is prevalent. OBJECTIVE: We hypothesized that zinc deficiency may potentiate disruption of endothelial cell integrity mediated by fatty acids and inflammatory cytokines by enhancing pathways that lead to apoptosis and up-regulation of caspase genes. DESIGN: Endothelial cells were maintained in low-serum medium or grown in culture media containing selected chelators, ie, diethylenetriaminepentaacetate or N,N,N', N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), with or without zinc supplementation. Subsequently, cells were treated with linoleic acid, tumor necrosis factor alpha (TNF-alpha), or both. We studied the effect of zinc deficiency and supplementation on the induction of apoptosis by measuring caspase-3 activity, cell binding of annexin V, and DNA fragmentation. RESULTS: Our results indicated that linoleic acid and TNF-alpha independently, but more markedly in concert, up-regulated caspase-3 activity and induced annexin V binding and DNA fragmentation. Zinc deficiency, especially when induced by TPEN, dramatically increased apoptotic cell death induced by cytokines and lipids compared with control cultures. Supplementation of low-serum- or chelator-treated endothelial cells with physiologic amounts of zinc caused a marked attenuation of apoptosis induced by linoleic acid and TNF-alpha. Morphologic changes of cells observed during zinc deficiency were prevented by zinc supplementation. Media supplementation with other divalent cations (eg, calcium and magnesium) did not mimic the protective role of zinc against apoptosis. CONCLUSIONS: Our data indicate that zinc is vital to vascular endothelial cell integrity, possibly by regulating signaling events to inhibit apoptotic cell death.     

Zinc nutrition and apoptosis of vascular endothelial cells: implications in atherosclerosis.

Little is known about the requirements and function of zinc in maintaining endothelial cell integrity, especially during stressful conditions, such as the inflammatory response in cardiovascular disease. There is evidence that zinc requirements of the vascular endothelium are increased during inflammatory conditions such as atherosclerosis, where apoptotic cell death is also prevalent. Apoptosis is a morphologically distinct mechanism of programmed cell death which involves the activation of a cell-intrinsic suicide program, and there is evidence that factors such as inflammatory cytokines (e.g., tumor necrosis factor [TNF]) and pure or oxidized lipids are necessary to induce the cell death pathway. Because of its constant exposure to blood components, including prooxidants, diet-derived fats, and their derivatives, the endothelium is very susceptible to oxidative stress and to apoptotic injury mediated by blood lipid components, prooxidants, and cytokines. Thus, it is likely that the cellular lipid environment, primarily polyunsaturated fatty acids, can potentiate the overall endothelial cell injury by increasing cellular oxidative stress and cytokine release in proximity to the endothelium, which then could further induce apoptosis and disrupt endothelial barrier function. Our data suggest that zinc deficiency exacerbates the detrimental effects of specific fatty acids (e.g., linoleic acid) and inflammatory cytokines, such as TNF, on vascular endothelial functions. We propose that a major mechanism of zinc protection against disruption of endothelial cell integrity during inflammatory conditions, is by the ability of zinc to inhibit the pathways of signal transduction leading to apoptosis and especially mechanisms that lead to upregulation of caspase genes.     

锌对体外培养小鼠胎鼠前肢骨发育的影响

目的 研究锌对骨发育的影响。方法 用一次通气旋转装置,在缺锌的基础培养基和分别加入45、70和120μmol／LZn^2 的培养基中对16d孕龄胎鼠前肢进行培养。结果 培养基中缺锌和在培养基中补充Zn^2 至Zn^2 浓度120μmol／L时,骨组织中骨钙素(OC)和^45Ca含量减少,碱性磷酸酶(AKP))活性显著降低;当在培养基中补充Zn^2 至Zn^2 浓度45、70μmol/L时OC合成量、骨组织对钙的吸收及AKP活性显著增加。培养液中缺锌及锌浓度为120μmol／L时,X线显示,长骨长度和密度与其自身对照相比,长骨长度较短,骨密度降低;当培养液中Zn^2 浓度分别为45和70μmol／L时,与其自身对照相比,长骨长度及其骨密度有所增加。组织学分析显示,培养液中缺锌及锌浓度为120μmol／L时,镜下可见骨细胞死亡,基质丢失;当培养液中Zn^2 浓度分别为45和70μmol／L时,骨细胞增殖、分化活跃,类骨质的分泌与合成增加。结论 适量补锌能促进骨组织形成及发育,锌缺乏或过量时均可影响骨组织正常生长发育及代谢。     

Zinc deficiency increases the osmotic fragility of rat erythrocytes

Zinc deficiency in rats causes increased osmotic fragility of their erythrocytes. This study was designed to determine the relationship of food intake and dietary sulfur amino acid level to the effect of low zinc status on fragility. Immature rats were fed for a 3-wk period a low zinc diet (less than 1 mg/kg) based on isolated soybean protein or a similar control diet (100 mg Zn/kg diet) supplied either ad libitum or by pair feeding. Fragility was measured by the degree of hemolysis in hypotonic saline solutions. In the first experiment, zinc deficiency resulted in higher fragility than in ad libitum controls; pair-fed controls were intermediate and not different from either. Experiment 2 included two levels of methionine, 0.4 and 0.9%, and two of zinc, 0 and 100 mg Zn/kg diet. At the 0.4%, but not at the 0.9% methionine level, hemolysis of red blood cells from the zinc-deficient rats was significantly greater than those from either pair-fed or ad libitum controls. Repletion for 1 or 2 d completely alleviated the increased fragility, but in vitro addition of zinc had no effect. Restricted intake of the zinc-adequate diet reversed the fragility within 1 d as readily as did ad libitum intake. Thus, the osmotic fragility induced by zinc deficiency was prevented by high sulfur amino acid intake and was readily reversed by dietary zinc. It is postulated that extracellular or membrane-bound zinc protects a component of the membrane that is essential to its function, and that reversal of the defect requires an in vivo metabolic process.     

锌缺乏和锌过量对培养长骨细胞周期和细胞凋亡的影响

目的　探讨锌缺乏和锌过量对培养长骨细胞周期和细胞凋亡的影响 ,为阐明锌对骨骼的生长发育影响的机制提供科学依据。方法　用小鼠胚胎长骨体外培养 ,采用流式细胞仪研究细胞周期各个时相所占比例和凋亡细胞百分比 ,进行电镜分析 ,观察细胞的超微结构。结果　缺锌可使细胞周期停滞在 G0 /G1期 ,培养 2 d,缺锌组 G0 /G1期细胞所占百分比 (82 .3% )高于对照组(76.9% ) ,G2 /M期细胞所占百分比 (3.4% )低于对照组 (8.9% ) ;培养 4d,缺锌组 G0 /G1期细胞所占百分比 (88.6% )明显高于对照组 (78.4% ) ,S期细胞所占百分比 (4.3% )明显低于对照组 (1 1 .0 % ) ;缺锌可使凋亡细胞数目增加。电镜观察 ,缺锌组可见大量的凋亡细胞 ,而高锌组则有大量的坏死细胞。结论　锌缺乏可改变培养长骨的细胞周期 ,诱导细胞凋亡 ,但锌过量对细胞周期和细胞凋亡没有影响。     

Accumulation and regulation of zinc in Daphnia magna: links with homeostasis and toxicity

Zinc accumulation in Daphnia magna was investigated, and the results were linked to the previously established optimal concentration range for zinc and D. magna. It was observed that organisms cultured in this optimal range (300-600 microg Zn/L) contained 212 +/- 57 to 254 +/- 79 microg Zn/g dry weight. Lower and higher zinc contents were obtained after acclimation to previously established culture concentrations inducing deficiency and toxicity, respectively. The calculation of bioconcentration factors indicated that zinc was actively regulated, at least up to a concentration of 600 microg Zn/L. Zinc uptake and elimination are rapid processes; major increases and decreases in body content occurred within 1 day. Zinc concentrations in daphnids exposed to 600 microg Zn/L fluctuated with 2- to 3-day intervals, suggesting a role of molting in the regulation and elimination of zinc.     

Zinc transporters in the rat mammary gland respond to marginal zinc and vitamin A intakes during lactation

Marginal intake of zinc and vitamin A is common during lactation and a deficiency of one micronutrient can result in a secondary deficiency of the other. However, the resistance of milk zinc (Zn) concentration to changes in dietary Zn or vitamin A indicates tight regulation of mammary gland Zn transport. Although several mammalian proteins have been identified and implicated in Zn transport, the mechanisms responsible for mammary gland Zn transport and their regulation by dietary Zn and vitamin A are unknown. In this study, we identified mammary gland Zn transporters and determined effects of marginal Zn and vitamin A intakes on their levels. Rats were fed a control [25 mg Zn/kg, 4 retinol equivalents (RE)/g], a low Zn (10 mg Zn/kg), a low vitamin A (0.4 RE/g), or a low Zn (10 mg Zn/kg) and vitamin A (0.4 RE/g) diet throughout lactation. ZnT-1, ZnT-2 and ZnT-4 were identified in the mammary gland and localized to the serosal membrane (ZnT-1) or intracellularly (ZnT-2 and ZnT-4) by immunostaining. Rats fed a low Zn or low vitamin A diet had lower ZnT-1 protein and higher ZnT-4 mRNA expression and protein levels compared with controls. There was a significant interaction between dietary Zn and vitamin A on zinc transporter mRNA expression and protein levels. Although total mammary gland Zn was not affected, mammary gland metallothionein levels were lower in rats fed low Zn and higher in rats fed low vitamin A, suggesting different mechanisms regulating zinc transporter levels. These results indicate that milk Zn level is maintained through coordinated regulation of mammary gland zinc transporters and documents an effect of vitamin A on zinc homeostasis at the molecular level during lactation.     

锌缺乏对培养小鼠胚胎长骨骺板c-fos基因转录和表达的影响

采用体外培养方法研究锌缺乏对小鼠胚胎长骨骺板ｃ－ｆｏｓ基因（ｃ－ｆｏｓ）转录与表达的影响。将孕龄１６天的小鼠胚胎长骨体外培养４８小时（培养基为ＧＢＪｂ）,通过免疫组化及原位杂交方法观察培养长骨ｃ－ｆｏｓｌ转录与表达的变化,并用图像分析系统对结果进行分析。根据培养基的锌浓度将实验分为对照组,缺锌组,缺锌补锌组,锌刺激量组。     

Subject Index to Volume 10

Reduced food consumption is a major manifestation of zinc (Zn) deficiency. Many manifestations of Zn deficiency are complications of anorexia nervosa and bulimia nervosa. We evaluated serum and 24-hour urinary Zn values in 12 healthy volunteers and 33 eating disorder patients before and after hospitalization which included either Zn supplementation (75 mg Zn/day) or placebo. Bulimics had depressed serum Zn concentrations (p < 0.025). Admission urinary Zn was lower in bulimics (258 +/? 44 micrograms/day), and significantly depressed in anorexics (196 +/? 36 micrograms/day, p < 0.005) vs controls (376 +/? 45 micrograms/day). During hospitalization, serum Zn concentrations increased in all supplemented patients vs no change with placebo. Urinary Zn excretion increased in supplemented bulimics (p < 0.001) and placebo (p < 0.05). Urinary Zn excretion markedly increased in supplemented anorexics (179 +/? 65 to 1052 +/? 242 micrograms/day); however, placebo values fell or remained unacceptably low (admission 208 +/? 48 micrograms/day; discharge 160 +/? 17 micrograms/day). By dietary history, controls consumed the Recommended Dietary Allowance (RDA) for Zn (11.95 +/? 1.25 mg/day); anorexics 6.46 +/? 1.14 mg/day; and bulimics 8.93 +/? 1.29 mg/day. We suggest that Zn deficiency may act as a “sustaining” factor for abnormal eating behavior in certain eating disorder patients.     

Zinc status before and after zinc supplementation of eating disorder patients.

Reduced food consumption is a major manifestation of zinc (Zn) deficiency. Many manifestations of Zn deficiency are complications of anorexia nervosa and bulimia nervosa. We evaluated serum and 24-hour urinary Zn values in 12 healthy volunteers and 33 eating disorder patients before and after hospitalization which included either Zn supplementation (75 mg Zn/day) or placebo. Bulimics had depressed serum Zn concentrations (p < 0.025). Admission urinary Zn was lower in bulimics (258 +/- 44 micrograms/day), and significantly depressed in anorexics (196 +/- 36 micrograms/day, p < 0.005) vs controls (376 +/- 45 micrograms/day). During hospitalization, serum Zn concentrations increased in all supplemented patients vs no change with placebo. Urinary Zn excretion increased in supplemented bulimics (p < 0.001) and placebo (p < 0.05). Urinary Zn excretion markedly increased in supplemented anorexics (179 +/- 65 to 1052 +/- 242 micrograms/day); however, placebo values fell or remained unacceptably low (admission 208 +/- 48 micrograms/day; discharge 160 +/- 17 micrograms/day). By dietary history, controls consumed the Recommended Dietary Allowance (RDA) for Zn (11.95 +/- 1.25 mg/day); anorexics 6.46 +/- 1.14 mg/day; and bulimics 8.93 +/- 1.29 mg/day. We suggest that Zn deficiency may act as a "sustaining" factor for abnormal eating behavior in certain eating disorder patients.     

Zinc deprivation impairs growth factor-stimulated calcium influx into murine 3T3 cells associated with decreased cell proliferation

Zinc plays a critical role in growth, a process that depends primarily on cell proliferation. Murine fibroblasts, Swiss 3T3 cells, were used to explore the hypothesis that a critical role of zinc in cell proliferation relates to its function in calcium influx. Cells were deprived of zinc by an impermeant chelator, diethylenetriaminepentaacetate (0.6 mmol/L), and low-calcium status was achieved by using a low- (<5 μmol/L) calcium medium. Cells were stimulated by a composite of growth factors (GF): platelet-derived GF, insulin-like GF-I, and epidermal GF. GF stimulation of cell proliferation was assessed by the incorporation of tritiated thymidine and calcium influx by the increase in fluorescence of cells loaded with Fluo-4. Proliferation was dependent on both zinc and calcium and they interacted in this process. GF stimulated an immediate sharp increase in intracellular calcium, indicative of internal calcium release, which peaked within 1 min and decreased to an elevated plateau, a pattern typical of a store-operated calcium channel. The sustained calcium influx of zinc-deprived cells was markedly lower than that of supplemented cells. Verapamil, a calcium channel blocker, also depressed both cell proliferation and calcium influx. In summary, zinc deficiency impaired GF-stimulated calcium influx into murine fibroblasts in association with decreased cell proliferation.     

Accumulation and Regulation of Zinc in Daphnia magna: Links with Homeostasis and Toxicity

Zinc accumulation in Daphnia magna was investigated, and the results were linked to the previously established optimal concentration range for zinc and D. magna. It was observed that organisms cultured in this optimal range (300–600 μg Zn/L) contained 212 ± 57 to 254 ± 79 μg Zn/g dry weight. Lower and higher zinc contents were obtained after acclimation to previously established culture concentrations inducing deficiency and toxicity, respectively. The calculation of bioconcentration factors indicated that zinc was actively regulated, at least up to a concentration of 600 μg Zn/L. Zinc uptake and elimination are rapid processes; major increases and decreases in body content occurred within 1 day. Zinc concentrations in daphnids exposed to 600 μg Zn/L fluctuated with 2- to 3-day intervals, suggesting a role of molting in the regulation and elimination of zinc. Received: 12 November 2001/Accepted: 15 April 2002