Zinc deficiency induces oxidative DNA damage and increases p53 expression in human lung fibroblasts

Poor zinc nutrition may be an important risk factor in oxidant release and the development of DNA damage and cancer. Approximately 10% of the United States population ingests <50% of the recommended daily allowance for zinc, a cofactor in proteins involved in antioxidant defenses, electron transport, DNA repair and p53 protein expression. This study examined the effects of zinc deficiency on oxidative stress, DNA damage and the expression of DNA repair enzymes in primary human lung fibroblasts by the use of DNA microarrays and functional assays. Cellular zinc was depleted by 1) growing cells in a zinc-deficient medium and 2) exposuring cells to an intracellular zinc chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine. Array data revealed upregulation of genes involved in oxidative stress and DNA damage/repair and downregulation of other DNA repair genes. Zinc deficiency in cells caused an increase in oxidant production (dichlorofluoroscein fluorescence) and a significant induction of single-strand breaks (Comet assay) and p53 protein expression (Western blot analysis). Thus, zinc deficiency not only caused oxidative stress and DNA damage, but also compromised the cells' ability to repair this damage. Zinc adequacy appears to be necessary for maintaining DNA integrity and may be important in the prevention of DNA damage and cancer.     

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: mechanisms of host defense

Zinc deficiency in humans decreases the activity of serum thymulin (a thymic hormone), which is required for maturation of T-helper cells. T-helper 1 (Th(1)) cytokines are decreased but T-helper 2 (Th(2)) cytokines are not affected by zinc deficiency in humans. This shift of Th(1) to Th(2) function results in cell-mediated immune dysfunction. Because IL-2 production (Th(1) cytokine) is decreased, this leads to decreased activities of natural-killer cell and T cytolytic cells, which are involved in killing viruses, bacteria, and tumor cells. In humans, zinc deficiency may decrease the generation of new CD4+ T cells from the thymus. In cell culture studies (HUT-78, a Th(0) human malignant lymphoblastoid cell line), as a result of zinc deficiency, nuclear factor-kappaB (NF-kappaB) activation, phosphorylation of IkappaB, and binding of NF-kappaB to DNA are decreased and this results in decreased Th(1) cytokine production. In another study, zinc supplementation to humans decreased the gene expression and production of pro-inflammatory cytokines and decreased oxidative stress markers. In HL-60 cells (a human pro-myelocytic leukemia cell line), zinc deficiency increased the levels of TNF-alpha, IL-1beta, and IL-8 cytokines and mRNA. In these cells, zinc induced A20, a zinc finger protein that inhibited NF-kappaB activation via tumor necrosis factor receptor associated factor pathway, and this decreased gene expression of pro-inflammatory cytokines and oxidative stress markers. We conclude that zinc has an important role in cell-mediated immune functions and also functions as antiinflammatory and antioxidant agent.     

The p53 gene and protein in 2005: new results, promising opportunities

The p53 gene and protein in 2005: new results, promising opportunities. The p53 gene is one of the most important genes which is involved in the regulation of cell division and tumor formation. The normal function of the p53 protein is to arrest the cell division and to turn the cell towards apoptosis in the case of cell stress and DNA damage, thereby to protect the integrity of the genome. Several p53 gene mutations that have function in carcinogenesis have been found in various tumors, including gastrointestinal carcinomas. The loss of p53 response plays an important role in the malignantly transformed cell proliferation. Promising experiments try to substitute the lost functions of the p53 gene. With their help new anti-tumor therapeutic methods should be developed.     

Zinc Deficiency Induces Vascular Pro-Inflammatory Parameters Associated with NF-κB and PPAR Signaling

Objectives: Marginal intake of dietary zinc can be associated with increased risk of cardiovascular diseases. In the current study we hypothesized that vascular dysfunction and associated inflammatory events are activated during a zinc deficient state.

Design: We tested this hypothesis using both vascular endothelial cells and mice lacking the functional LDL-receptor gene.

Results: Zinc deficiency increased oxidative stress and NF-κB DNA binding activity, and induced COX-2 and E-selectin gene expression, as well as monocyte adhesion in cultured endothelial cells. The NF-κB inhibitor CAPE significantly reduced the zinc deficiency-induced COX-2 expression, suggesting regulation through NF-κB signaling. PPAR can inhibit NF-κB signaling, and our previous data have shown that PPAR transactivation activity requires adequate zinc. Zinc deficiency down-regulated PPARα expression in cultured endothelial cells. Furthermore, the PPARγ agonist rosiglitazone was unable to inhibit the adhesion of monocytes to endothelial cells during zinc deficiency, an event which could be reversed by zinc supplementation. Our in vivo data support the importance of PPAR dysregulation during zinc deficiency. For example, rosiglitazone induced inflammatory genes (e.g., MCP-1) only during zinc deficiency, and adequate zinc was required for rosiglitazone to down-regulate pro-inflammatory markers such as iNOS. In addition, rosiglitazone increased IκBα protein expression only in zinc adequate mice. Finally, plasma data from LDL-R-deficient mice suggest an overall pro-inflammatory environment during zinc deficiency and support the concept that zinc is required for proper anti-inflammatory or protective functions of PPAR.

Conclusions: These studies suggest that zinc nutrition can markedly modulate mechanisms of the pathology of inflammatory diseases such as atherosclerosis.     

Zinc Status Alters Growth and Oxidative Stress Responses in Rat Hepatoma Cells

Zinc deficiency and excess influence cellular homeostasis and are believed to modulate apoptosis. Zinc also regulates cell growth and proliferation. Understanding of the role of zinc in the mechanisms associated with these changes is limited because of its diverse, complex, and cell-specific effects. Therefore, we investigated the oxidative stress responses and the underlying molecular mechanisms associated with the disruption of intracellular zinc homeostasis in H4IIE rat hepatoma cells. We found that zinc excess (100 μM) and DTPA (diethylenetriaminepentaacetic acid; 50–100 μM) induced zinc deficiency both generate reactive oxygen species (ROS) and decrease viability in H4IIE cells. However, cotreatment with the antioxidant, N-acetyl-L-cysteine (NAC) both reduced ROS production and protected cells from death. We additionally observed an increase in Bax mRNA and cytochrome c release from the mitochondria in DTPA-treated cells and an elevated expression of Fas/Fas ligand mRNA with zinc treatment. Both treatments increased p53 and MdM2 protein concentrations along with caspase 3/7 activity. These results suggest that zinc deficiency stimulates mitochondrial-dependent apoptosis whereas zinc activates the extrinsic-apoptotic pathway. Both decreasing and increasing cellular zinc concentrations modulate ROS mediated apoptosis and warrant further research on zinc mediated cancer chemoprevention in this and other cancer cell lines.     

Mechanisms of benzene-induced hematotoxicity and leukemogenicity: cDNA microarray analyses using mouse bone marrow tissue

Although the mechanisms underlying benzene-induced toxicity and leukemogenicity are not yet fully understood, they are likely to be complicated by various pathways, including those of metabolism, growth factor regulation, oxidative stress, DNA damage, cell cycle regulation, and programmed cell death. With this as a background, we performed cDNA microarray analyses on mouse bone marrow tissue during and after a 2-week benzene exposure by inhalation. Our goal was to clarify the mechanisms underlying the hematotoxicity and leukemogenicity induced by benzene at the level of altered multigene expression. Because a few researchers have postulated that the cell cycle regulation mediated by p53 is a critical event for benzene-induced hematotoxicity, the present study was carried out using p53-knockout (KO) mice and C57BL/6 mice. On the basis of the results of large-scale gene expression studies, we conclude the following: (a) Benzene induces DNA damage in cells at any phase of the cell cycle through myeloperoxidase and in the redox cycle, resulting in p53 expression through Raf-1 and cyclin D-interacting myb-like protein 1. (b) For G1/S cell cycle arrest, the p53-mediated pathway through p21 is involved, as well as the pRb gene-mediated pathway. (c) Alteration of cyclin G1 and Wee-1 kinase genes may be related to the G2/M arrest induced by benzene exposure. (d) DNA repair genes such as Rad50 and Rad51 are markedly downregulated in p53-KO mice. (e) p53-mediated caspase 11 activation, aside from p53-mediated Bax gene induction, may be an important pathway for cellular apoptosis after benzene exposure. Our results strongly suggest that the dysfunction of the p53 gene, possibly caused by strong and repeated genetic and epigenetic effects of benzene on candidate leukemia cells, may induce fatal problems such as those of cell cycle checkpoint, apoptosis, and the DNA repair system, finally resulting in hemopoietic malignancies. Our cDNA microarray data provide valuable information for future investigations of the mechanisms underlying the toxicity and leukemogenicity of benzene.     

Folate deficiency in vitro induces uracil misincorporation and DNA hypomethylation and inhibits DNA excision repair in immortalized normal human colon epithelial cells

Epidemiological studies have indicated that folic acid protects against a variety of cancers, particularly cancer of the colorectum. Folate is essential for efficient DNA synthesis and repair. Moreover, folate can affect cellular S-adenosylmethionine levels, which regulate DNA methylation and control gene expression. We have investigated the mechanisms through which folate affects DNA stability in immortalized normal human colonocytes (HCEC). DNA strand breakage, uracil misincorporation, and DNA repair, in response to oxidative and alkylation damage, were determined in folate-sufficient and folate-deficient colonocytes by single cell gel electrophoresis. In addition, methyl incorporation into genomic DNA was measured using the bacterial enzyme Sss1 methylase. Cultured human colonocyte DNA contained endogenous strand breaks and uracil. Folate deficiency significantly increased strand breakage and uracil misincorporation in these cells. This negative effect on DNA stability was concentration dependent at levels usually found in human plasma (1-10 ng/ml). DNA methylation was decreased in HCEC grown in the absence of folate. Conversely, hypomethylation was not concentration dependent. Folate deficiency impaired the ability of HCEC to repair oxidative and alkylation damage. These results demonstrate that folic acid modulates DNA repair, DNA strand breakage, and uracil misincorporation in immortalized human colonocytes and that folate deficiency substantially decreases DNA stability in these cells.     

环境致癌剂与p53基因突变

p5 3基因是至今为止发现的与人类肿瘤相关性最高的基因。它不仅作为抑癌基因发挥作用 ,还参与细胞周期调控、DNA损伤与修复、基因转录及细胞凋亡等多个过程。肿瘤病因学的研究表明 ,6 0 %～ 90 %的人类肿瘤是由环境中的化学致癌物引起。环境中的化学致癌物或前致癌物可以造成p5 3基因突变及蛋白表达的改变 ,且p5 3的突变形式也随着致癌物和肿瘤的类型不同而具有特征性的差异 ,相关研究已成为近年来肿瘤分子生物学的热点。本文综述了苯并 [a]芘、烟草、亚硝胺、黄曲霉毒素和氯乙烯等几种环境中存在的主要化学致癌物质的致癌效应以及对p5 3基因和P5 3蛋白的影响。     

氯乙烯致大鼠DNA损伤与DNA损伤修复酶和p53蛋白的表达

目的研究氯乙烯（VC）对大鼠原代肝细胞DNA的损伤作用,及对DNA损伤修复酶（rMSH2和XPD）和抑癌蛋白p53表达的影响;探索VC所致DNA损伤的修复和调控机制。方法大鼠腹腔注射VC,隔日染毒,染毒剂量分别为5,10和20mg／kg。单细胞凝胶电泳测肝细胞DNA损伤,免疫组化法测肝脏DNA损伤修复酶的表达。结果彗星细胞数目随染毒剂量增加而增加,彗星发生率与VC染毒剂量问存在明显的相关关系。rMSH2表达随染毒剂量增加而减少．XPD和p53的表达随染毒剂量增加而增加。VC致DNA损伤与XPD表达具有相关关系。结论VC可导致肝细胞DNA发生损伤,且存在剂量一反应关系;DNA损伤修复酶和p53蛋白参与修复VC所致的DNA损伤。     

900 MHz电磁辐射对人胚肺细胞DNA及p53基因蛋白表达的影响

目的 研究900 MHz电磁辐射对人胚肺细胞DNA及p53基因蛋白表达的影响.方法 用900 MHz电磁波辐照细胞.单细胞凝胶电泳实验分为1、2、5、8 mW/cm2 4个暴露组,并设阴性对照组(0mW/cm2)和阳性对照组(0.1 mmol/L重铬酸钾处理),每组2个平行样,暴露时间均为1 h,检测拖尾率和DNA迁移长度.Western Blot实验分为1、2、5、8 mW/cm2 4个暴露组,并设阴性对照组(0 mW/cm2)和阳性对照组(淋巴瘤细胞株Raji),暴露时间均为12 h,检测电磁辐射对p53基因蛋白表达的影响.结果 与阴性对照组(0 mW/cm2)比较,各暴露组的拖尾率和DNA迁移长度差异无统计学意义(P＞0.05).各暴露组和阴性对照组(0 mW/cm2)的细胞中p53蛋白表达为阴性,而阳性对照组检测到了p53蛋白的表达.结论 本实验未观察到900 MHz电磁辐射对人胚肺细胞DNA及p53基因蛋白表达有影响.     

Expression of the p53 tumor suppressor gene is up-regulated by depletion of intracellular zinc in HepG2 cells

Expression and activation of the p53 tumor suppressor protein are modulated by various cellular stimuli. The objective of this work was to examine the influence of zinc depletion on the expression of p53 in HepG2 cells. Two different low Zn (ZD) media, Zn-free Opti-MEM and a ZD medium containing Chelex-100 treated serum, were used to deplete cellular zinc over one passage. Cellular zinc levels of ZD cells were significantly lower than in their controls in both the Opti-MEM and Chelex studies. p53 mRNA abundance was 187% higher in ZD Opti-MEM cells and >100% higher in ZD Chelex cells compared with their respective controls. To examine whether the effects were specific to zinc depletion, a third, zinc-replenished group (ZDA) was included in the Opti-MEM study in which cells were cultured in ZD media for nearly one passage before a change was made to zinc-adequate (ZA) medium for the last 24 h. Zinc levels in the ZDA cells were significantly higher than in ZD cells, and p53 mRNA abundance was normalized to control levels. Nuclear p53 protein levels were >100% higher in the ZD Opti-MEM cells than in ZA cells. Interestingly, the ZDA Opti-MEM cells had significantly lower levels of nuclear p53 protein than both the ZA and ZD cells. These data suggest that expression of p53, a critical component in the maintenance of genomic stability, may be affected by reductions in cellular zinc.