Zinc ionophores isolated from Terminalia bellirica fruit rind extract protect against cardiomyocyte hypoxia/reoxygenation injury.

The study aimed to isolate and characterize zinc ionophores from Terminalia bellirica fruit using a liposome assay and test its utility in H9c2 rat cardiomyoblasts cells subjected to hypoxia/reoxygenation. Ethyl acetate extract that exhibited zinc ionophore activity was resolved to yield three polyphenols that were characterized as epicatechin-3-gallate (ECG), epigallocatechin-3-gallate (EGCG) and epigallocatechin (EGC) by nuclear magnetic resonance and electrospray ionization-mass spectra. The polyphenols enhanced the uptake of zinc into the liposomes and increased FluoZin-3 fluorescence. These polyphenols in the presence of 10 µM ZnCl2 enhanced the zinc import into H9c2 cells, whose intracellular zinc levels were otherwise lowered upon hypoxia/reoxygenation. EGCG proved to be more potent than ECG, which indeed was more effective than EGC in improving cellular zinc levels and in attenuating the apoptosis of H9c2 cells after hypoxia/reoxygenation injury. The polyphenols required zinc for anti-apoptotic effect. The cardioprotective effect is indeed due to enhanced zinc uptake mediated by these polyphenols.

Zinc-dependent changes in oxidative and endoplasmic reticulum stress during cardiomyocyte hypoxia/reoxygenation.

Myocardial zinc dyshomeostasis is associated with caspase-3 activation, ErbB2 degradation and apoptosis during hypoxia/reoxygenation. Zinc pyrithione replenishes intracellular zinc, suppresses caspase-3, augments ErbB2 levels and improves cell survival. We hypothesize that zinc is capable of modulating redox and endoplasmic reticulum (ER) stress in the setting of cardiomyocyte hypoxia-reoxygenation. Hypoxia/reoxygenation lowered intracellular zinc, increased ER as well as oxidative stress in H9c2 cells, both of which were effectively attenuated by zinc supplementation. Silencing of gp91phox attenuated oxidative and ER stress, decreased caspase-3 activation and improved cell survival. Mimicking the oxidative insult using 50 µM H2O2 increased the caspase-3 activity that correlated with decreased ErbB2 levels, concomitant with augmented ER stress. N-acetyl cysteine (NAC) administration completely suppressed ER stress as well as caspase-3 activity. Zinc depletion using TPEN also resulted in lowered ErbB2 and increased apoptosis, along with NOX2 mRNA upregulation, increased oxidative and ER stress. Repletion with zinc suppressed NOX2 mRNA, lowered oxidative as well as ER stress and decreased cell death. These results suggest that zinc dyshomeostasis, along with oxidative stress contribute to the unfolded protein response during myocardial H/R and that zinc replenishment corrects zinc homeostasis, alleviates associated stress and improves cardiomyocyte survival.

Dietary phytate lowers K-ras mutational frequency, decreases DNA-adduct and hydroxyl radical formation in azoxymethane-induced colon cancer.

OBJECTIVES: Dietary phytate is known to protect against azoxymethane (AOM)-induced preneoplastic lesions. The present study was designed to determine whether dietary phytate affects mutation frequency in colon epithelial cells challenged with azoxymethane in vivo, through lowering the formation of O6-methyl guanosine (O6-MeG) and 8-hydroxy deoxyguanosine (8-OHdG) adducts. MATERIALS AND METHODS: We used Fisher F344 rats induced with AOM for 20 weeks and undertook 1% or 2% phytate supplementation for subsequent 16 weeks to monitor the mutation frequencies of one of the candidate genes, K-ras, along with DNA adduct load. RESULTS: Dietary phytate significantly suppressed aberrant crypt foci formation and effectively inhibited colon tumor formation in a dose-dependent manner. DNA sequencing results demonstrated that 60% of the colon tumors from AOM-treated and control diet fed animals showed GGT to GAT transition and 40% of the tumors showed GGT to GTT transversion at codon 12, along with 18% of the tumors showing GGC to CGC transversion at codon 13. Phytate supplementation at 1 and 2% lowered the frequency of GGT > GAT to 30 and 10%, respectively. Phytate supplementation also nullified the codon 13 mutations. No mutations were observed at codon 61 in any of the experimental groups. CONCLUSION: The lowered frequency of K-ras mutations correlated with decreased formation of hydroxyl radicals, O5-meG and 8-OH-dG levels in phytate-supplemented animals with lowered tumor burden.

Comparative Response of Cardiomyocyte ZIPs and ZnTs to Extracellular Zinc and TPEN.

Intracellular zinc concentrations are tightly regulated by the coordinated regulation of ZIPs and ZnTs. Very little is known about the regulation of these transporters in cardiomyocytes, in response to extracellular zinc. Adult rat cardiomyocytes express ZnTs 1, 2, 5, and 9, in addition to ZIPs 1, 2, 3, 6, 7, 9, 10, 11, 13, and 14. We have determined the intracellular free zinc levels using Zinpyr-1 fluorescence and studied response of ZIP and ZnT mRNA by real-time PCR to the changes in extracellular zinc and TPEN in adult rat ventricular myocytes. TPEN downregulated ZnT1, ZnT2, and ZIP11 mRNAs but upregulated ZnT5, ZIP2, ZIP7, ZIP10, ZIP13, and ZIP14 mRNAs. Zinc supplementation upregulated ZnT1, ZnT2 mRNA but downregulated ZnT5, ZIP1, ZIP2, ZIP3, ZIP7, ZIP9, and ZIP10 mRNA. The negative regulation of ZIPs by zinc excess can be explained in terms of zinc homeostasis as these transporters may act to protect cells from zinc over accumulation by reducing zinc influx when the extracellular concentration of zinc is high. Similarly, the ZnT expression appears to be regulated to avoid loss of zinc from the intracellular milieu, under zinc-deficient conditions.

Intracellular zinc status influences cisplatin-induced endothelial permeability through modulation of PKCα, NF-κB and ICAM-1 expression.

Platinum-based chemotherapeutic regimen induces vascular dysfunction. Action of cisplatin on endothelial cells is mediated by protein kinase C (PKC-α), which further activates nuclear factor-κB (NF-κB) and induces canonical transient receptor potential channel (TRPC1) and intercellular adhesion molecule (ICAM-1) expression. Increased ICAM-1 contributes to hyperadhesion of monocytes and endothelial dysfunction. PKC-α is also involved in phosphorylation of TRPC1, resulting in store-operated calcium entry (SOCE) and further activation of NF-κB. Although the role of altered intracellular zinc status is not known in cisplatin-induced vascular dysfunction, because of the ability of zinc to modulate PKC-α, NF-κB activity, we hypothesized that zinc can ameliorate the extent of endothelial dysfunction induced by cisplatin. Human umbilical vein endothelial cells treated with cisplatin (8.0µg/ml) showed lowered intracellular free zinc, concomitant with enhanced activation of PKC-α, NF-κΒ activation, TRPC1, SOCE and ICAM-1 levels. Zinc deficiency per se induced using membrane permeable chelator (TPEN) mimicked the cisplatin-induced PKC-α, NF-κB activation and ICAM-1 expression, but also activated Activator Protein-1 (AP-1). Zinc supplementation (2.0-10.0µM) to the endothelial cells during cisplatin treatment or TPEN-induced zinc deficiency suppressed PKC-α, NF-κB, TRPC1, SOCE activation and lowered the ICAM-1 expression. Zinc supplementation thereby effectively decreased the cisplatin-induced endothelial permeability and adherence of the activated endothelial cells to U937 monocytes.

Effect of vitamin supplementation on cisplatin-induced intestinal epithelial cell apoptosis in Wistar/NIN rats.

OBJECTIVE: Chemotherapeutic agents induce small intestinal mucositis that is characterized structurally by crypt loss and villus atrophy and functionally by absorptive and barrier impairments. We studied the effect of selected individual vitamins and multiple-vitamin mixture supplementation in modulating cisplatin-induced intestinal damage and apoptosis. METHODS: Thirty-six male Wistar/NIN rats 20 wk old and fed the control diet (AIN-93G) were randomly divided into six groups. Five groups were administered cisplatin (2.61 mg/kg of body weight) once a week for 3 wk and were concomitantly provided the control diet or riboflavin, folate, α- tocopherol, or a multiple-vitamin mixture supplemented diet. The sixth group served as a control for cisplatin and received saline as the vehicle. Intestinal epithelial cell apoptosis was monitored by morphometry, M30 staining, DNA fragmentation, and caspase-3 activity. Functional and structural integrities were determined by measuring activities of alkaline phosphatase and lysine ala-dipeptidyl aminopeptidase and the villus height/crypt depth ratio. Oxidative burden was assessed as the formation of thiobarbituric acid-reactive substances and protein carbonyls. Plasma levels of selected vitamins were also measured. RESULTS: Cisplatin administration significantly increased intestinal apoptosis in the villus and crypt regions that correlated with increased oxidative damage, decreased Bcl-2/Bax, and compromised functional integrity. Riboflavin, folate, and the multiple-vitamin mixture supplementation attenuated the cisplatin-induced increase in apoptotic indices, with a decrease in oxidative burden, increased Bcl-2/Bax, and improved functional and structural integrities. The α-tocopherol supplementation, although effective in attenuating oxidative stress and improving functional integrity, failed to lower the apoptotic indices. CONCLUSIONS: Riboflavin, folate, and the multiple-vitamin supplementation proved to be more efficacious in attenuating the cisplatin-induced intestinal damage and associated changes in apoptosis.

Concurrent repletion of iron and zinc reduces intestinal oxidative damage in iron- and zinc-deficient rats.

AIM: To understand the interactions between iron and zinc during absorption in iron- and zinc-deficient rats, and their consequences on intestinal oxidant-antioxidant balance. METHODS: Twenty-four weanling Wistar-Kyoto rats fed an iron- and zinc-deficient diet (< 6.5 mg Fe and 4.0 mg Zn/kg diet) for 4 wk were randomly divided into three groups (n = 8, each) and orally gavaged with 4 mg iron, 3.3 mg zinc, or 4 mg iron + 3.3 mg zinc for 2 wk. At the last day of repletion, 3 h before the animals were sacrificed, they received either 37 mBq of (55)Fe or (65)Zn, to study their localization in the intestine, using microautoradiography. Hemoglobin, iron and zinc content in plasma and liver were measured as indicators of iron and zinc status. Duodenal sections were used for immunochemical staining of ferritin and metallothionein. Duodenal homogenates (mitochondrial and cytosolic fractions), were used to assess aconitase activity, oxidative stress, functional integrity and the response of antioxidant enzymes. RESULTS: Concurrent repletion of iron- and zinc-deficient rats showed reduced localization of these minerals compared to rats that were treated with iron or zinc alone; these data provide evidence for antagonistic interactions. This resulted in reduced formation of lipid and protein oxidation products and better functional integrity of the intestinal mucosa. Further, combined repletion lowered iron-associated aconitase activity and ferritin expression, but significantly elevated metallothionein and glutathione levels in the intestinal mucosa. The mechanism of interactions during combined supplementation and its subsequent effects appeared to be due to through modulation of cytosolic aconitase, which in turn influenced the labile iron pool and metallothionein levels, and hence reduced intestinal oxidative damage. CONCLUSION: Concurrent administration of iron and zinc corrects iron and zinc deficiency, and also reduces the intestinal oxidative damage associated with iron supplementation.