BioMetals (v.24, #1)

During the last decade, there have been concerted efforts to reduce beryllium (Be) exposure in the workplace and thereby reduce potential cases of this occupational lung disorder. Despite these efforts, it is estimated that there are at least one million Be-exposed individuals in the U.S. who are potentially at risk for developing chronic beryllium disease (CBD). Previously, we reviewed the current CBD literature and proposed that CBD represents a model interaction between innate and acquired immunity (Sawyer et al., Int Immunopharmacol 2:249–261, 2002). We closed this review with a section on “future directions” that identified key gaps in our understanding of the pathogenesis of CBD. In the intervening period, progress has been made to fill in some of these gaps, and the current review will provide an update on that progress. Based on recent findings, we provide a new hypothesis to explain how Be drives sustained chronic inflammation and granuloma formation in CBD leading to progressive compromised lung function in CBD patients. This paradigm has direct implications for our understanding of the development of an immune response to Be, but is also likely applicable to other immune-mediated lung diseases of known and unknown etiology.
Keywords: Beryllium; Chronic beryllium disease; Granuloma; Innate immunity; Acquired immunity

Does high serum iron level induce low bone mass in sickle cell anemia ? by Mir Sadat-Ali; Osama Sultan; Haifa Al-Turki; Abdulmohsen AlElq (19-22).
Iron overload is quite common in patients suffering from hemoglobinopathies causing arthropathies, endocrinal affection and neuropathies. Recently low bone mass was added to the list of complications. This study is conducted to find any correlation between serum iron level and low bone mass in sickle cell anemia (SCA). Patients ≥18 years of age with sickle cell anemia, who attended outpatient clinics or admitted to King Fahd University Hospital, Al Khobar, Saudi Arabia,between 1st September 2006 and August 2007 were the subjects of this study. Patients age and sex were documented and body mass index was calculated. Apart from routine hematological tests, serum ferritin, serum Iron level, total estradiol, testosterone level was done. Bone mineral density measurement was done using dual energy X-ray absorptiometry (DEXA) at upper femur and lumbar spine. The data of 100 patients was analyzed, 48 males and 52 females. The mean age was 27.5 ± 6.1 years. In 64 patients (32 males and 32 females) serum iron level was 319.35 μg/dl and the mean serum ferritin level in males and females was within the normal range. Sixty-eight percent of females and 71.8% of males patients in whom serum iron was high had lower bone mass P = < 0.001. Our study shows that SCA patients in whom serum iron level was higher than normal effected bone mass. Further studies are needed to confirm this as a cause of osteoporosis in SCA patients.
Keywords: Low bone mass; Serum iron level; Sickle cell anemia; Osteoporosis

Dietary zinc mediates inflammation and protects against wasting and metabolic derangement caused by sustained cigarette smoke exposure in mice by Carol J. Lang; Michelle Hansen; Eugene Roscioli; Jessica Jones; Chiara Murgia; Margaret Leigh Ackland; Peter Zalewski; Gary Anderson; Richard Ruffin (23-39).
In mouse asthma models, inflammation can be modulated by zinc (Zn). Given that appetite loss, muscle wasting and poor nutrition are features of chronic obstructive pulmonary disease (COPD) and that poor dietary Zn intake is in itself accompanied by growth retardation and appetite loss, we hypothesised that dietary Zn limitation would not only worsen airway inflammation but also exaggerate metabolic effects of cigarette smoke (CS) exposure in mice. Conversely, Zn supplementation would lessen inflammation. Mice were exposed to CS [2× 2RF, 3×/day; 15 min/cigarette] and fed diets containing 2, 20 or 140 mg/kg Zn ad libitum. Airway cells were collected by bronchoalveolar lavage (BAL). Plasma Zn was measured by fluorometric assay. Inflammatory, metabolic and Zn transport markers were measured by real-time RT-PCR. Mice fed low Zn diets had less plasma labile zinc (0–0.18 μM) than mice fed moderate (0.61–0.98 μM) or high (0.77–1.1 μM) Zn diets (SDs 0.1–0.4, n = 8–10). Smoke exposure increased plasma and BAL labile Zn (1.5–2.5 fold, P < 0.001), bronchoalveolar macrophages (2.0 fold, P < 0.0001) and MT-1 (1.5 fold), MIP-2 (2.3 fold) and MMP-12 (3.5 fold) mRNA. Zn supplementation reduced alveolar macrophage numbers by 62 and 52% in sham and smoke-exposed mice, respectively (Zn effect: P = 0.011). Gastrocnemius, soleus and tibialis anterior muscle mass were affected by both smoke and dietary Zn in the order of 3–7%. The 50–60% reduction in alveolar macrophages in Zn-supplemented mice supports our evolving hypothesis that Zn is an important anti-inflammatory mediator of airway inflammation. Restoring airway Zn levels through dietary supplementation may lessen the severity of lung inflammation when Zn intake is low.
Keywords: Zinc; Zinc transporters; Cigarette smoke; Chronic Obstructive Pulmonary Disease (COPD); Inflammation; Metabolic wasting

Quercetin, one of the most common dietary flavonols, was investigated in the presence of Cu(II) ions in methanolic solution in order to obtain some explanation on the mechanism interaction and its action against free radical-mediated damage. The spectroscopic studies (UV–VIS, IR, ESI–MS) were used to assess the extent to which it undergo complex formation through chelation or modification through oxidation. The reaction of quercetin with Cu(II) resulted in the formation of 1:1 metal–ligand complex (λmax = 436 nm) through the carbonyl oxygen and 3-OH group in C ring. Then quercetin is oxidized to the benzoquinone type products. The addition of EDTA destroyed the complex but did not regenerate the whole original spectrum of quercetin. From the other hand, the presence of EDTA inhibits formation of copper–quercetin complex and quercetin oxidation. The antioxidant activity of the Q + Cu solutions was evaluated by using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH∙) radical scavenging method and from an electrochemical point of view. The complex is much more effective as free radical scaveninger than the free flavonoid.
Keywords: Flavonoid; Quercetin; Copper complexation; Antioxidant properties

To understand how plants from the Fabaceae family maintain zinc (Zn) homeostasis, we have characterized the kinetics of three Zn transporting proteins from the ZIP family of divalent metal transporters in the model legume Medicago truncatula. Of six ZIP’s studied, MtZIP1, MtZIP5 and MtZIP6 were the only members from this family determined to transport Zn and were further characterized. MtZIP1 has a low affinity for Zn with a Km of 1 μM as compared to MtZIP5 and MtZIP6 that have a higher affinity for Zn with Km of 0.4 μM and 0.3 μM, respectively. Zn transport by MtZIP1 was more sensitive to inhibition by copper (Cu) concentrations than MtZIP5 and MtZIP6, because 3 μM Cu inhibited Zn transport by 80% in MtZIP1 while 5 μM Cu was required to achieve the same inhibition of Zn transport in MtZIP5 and MtZIP6. Cadmium (Cd) had a greater effect on the ability of MtZIP1 to transport Zn than MtZIP5 and MtZIP6, because at a concentration of 3 μM Cd, the Zn transport by MtZIP1 was inhibited 55% and the transport of Zn by MtZIP5 and MtZIP6 was inhibited by 20–30%. However, only MtZIP6 transported Cd at higher rates than those observed in the control plasmid pFL61, demonstrating a low affinity for Cd based on a Km of 57 μM. These results suggest that Medicago truncatula has both high and low affinity Zn transporters to maintain Zn homeostasis and that these transporters may function in different compartments within the plant.
Keywords: Zinc; Membrane transport; Kinetics; Cadmium; Copper; Medicago truncatula

Morphophysiological responses and programmed cell death induced by cadmium in Genipa americana L. (Rubiaceae) by Vânia L. Souza; Alex-Alan F. de Almeida; Stella G. C. Lima; Júlio C. de M. Cascardo; Delmira da C. Silva; Pedro A. O. Mangabeira; Fábio P. Gomes (59-71).
Cadmium (Cd) originating from atmospheric deposits, from industrial residues and from the application of phosphate fertilizers may accumulate in high concentrations in soil, water and food, thus becoming highly toxic to plants, animals and human beings. Once accumulated in an organism, Cd discharges and sets off a sequence of biochemical reactions and morphophysiological changes which may cause cell death in several tissues and organs. In order to test the hypothesis that Cd interferes in the metabolism of G. americana, a greenhouse experiment was conducted to measure eventual morphophysiological responses and cell death induced by Cd in this species. The plants were exposed to Cd concentrations ranging from 0 to 16 mg l−1, in a nutritive solution. In TUNEL reaction, it was shown that Cd caused morphological changes in the cell nucleus of root tip and leaf tissues, which are typical for apoptosis. Cadmium induced anatomical changes in roots and leaves, such as the lignification of cell walls in root tissues and leaf main vein. In addition, the leaf mesophyll showed increase of the intercellular spaces. On the other hand, Cd caused reductions in the net photosynthetic rate, stomatal conductance and leaf transpiration, while the maximum potential quantum efficiency of PS2 (Fv/Fm) was unchanged. Cadmium accumulated in the root system in high concentrations, with low translocation for the shoot, and promoted an increase of Ca and Zn levels in the roots and a decrease of K level in the leaves. High concentrations of Cd promoted morphophysiological changes and caused cell death in roots and leaves tissues of G. americana.
Keywords: Anatomy; Apoptosis; Heavy metal; Mineral nutrients; Photosynthesis

Differential responses of oat genotypes: oxidative stress provoked by aluminum by Luciane Belmonte Pereira; Cinthia Melazzo de A. Mazzanti; Denise Cargnelutti; Liana Verônica Rossato; Jamile F. Gonçalves; Nicéia Calgaroto; Valderi Dressler; Fernando T. Nicoloso; Luiz Carlos Federizzi; Vera M. Morsch; Maria R. C. Schetinger (73-83).
The phytotoxic effects of aluminum and the mechanisms of genetically-based Al tolerance have been widely investigated, as reported in many papers and reviews. However, investigations on many Al-sensitive and Al-resistant species demonstrate that Al phytotoxicity and Al-resistance mechanisms are extremely complex phenomena. The objective of the present study was to analyze the effects of aluminum on the activity of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX). Also was evaluated the lipid peroxidation, H2O2 content, levels of ascorbic acid (ASA), non-protein thiols (NPSH) and Al content in three genotypes of oat, Avena sativa L. (UFRGS 930598, UFRGS 17, and UFRGS 280). The genotypes were grown in different concentrations of Al ranging from 90 to 555 μM for 5 days. The antioxidant system was unable to overcome toxicity resulting in negative effects such as lipid peroxidation and H2O2 content in UFRGS 930598. The results showed that UFRGS 930598 was the most sensitive genotype. UFRGS 17 and UFRGS 280 were more resistant to Al toxicity. These results suggest that UFRGS 17 has mechanisms of external detoxification and UFRGS 280 has mechanisms of internal detoxification. The different behavior of enzymatic and non-enzymatic antioxidants of the genotypes showed that aluminum resistance in UFGRS 17 and UFRGS 280 may be related to oxidative stress.
Keywords: Avena sativa ; Superoxide dismutase; Hydrogen peroxide; Ascorbate peroxidase; Catalase; Aluminum content; Oxidative stress

Identification of new members within suites of amphiphilic marine siderophores by Julia M. Vraspir; Pamela D. Holt; Alison Butler (85-92).
Marine bacterial isolates Vibrio sp. HC0601C5 and Halomonas meridiana str. HC4321C1 were isolated off the coast of southern California and were found to produce an expanded suite of previously identified amphiphilic siderophores. Specifically two new members of the amphibactin family, amphibactins S and T, which have a C14:1 ω-7 fatty acid and a saturated C12 fatty acid, respectively, were produced by Vibrio sp. HC0601C5. These siderophores are produced in addition to a number of previously described amphibactins and are excreted into the culture supernatant. Two new members of the aquachelin family of siderophores, aquachelins I and J, which have an hydroxylated C12 fatty acid and a saturated C10 fatty acid, respectively, were produced by Halomonas meridiana str. HC4321C1. These four new siderophores are more hydrophilic than their previously reported relatives, aquachelins A–D and the amphibactin suite of siderophores.
Keywords: Marine siderophores; Amphiphilic siderophores; Amphibactins; Aquachelins

Following previous findings that cadmium (Cd) induces heme oxygenase-1 (HO1) gene expression in alfalfa seedling roots, we now show that the decreased glutathione (GSH) and ascorbic acid (AsA) contents, induction of HO-1 gene expression and its protein level by Cd was mimicked by a GSH depletor diethylmaleate (DEM). Meanwhile, above Cd- or DEM-induced decreased GSH content followed by HO-1 up-regulation could be strengthened or reversed differentially by the application of a selective inhibitor of GSH biosynthesis l-buthionine-sulfoximine (BSO), or exogenous GSH and AsA, respectively. The antioxidative behavior of HO-1 induction was further confirmed by histochemical staining for the detection of loss of membrane integrity in a short period of treatment time. Additionally, the induction of HO-1 transcript was inhibited by the transcriptional inhibitor actinomycin D (ActD) or protein synthesis inhibitor cycloheximide (CX, especially). In contrast, the level of HO-2 transcript did not change upon various treatments. Together, above results suggested that Cd-induced up-regulation of HO-1 gene expression is associated with GSH depletion, which is at least existing transcriptional regulation level, thus leading to enhanced antioxidative capability transiently.
Keywords: Alfalfa seedling roots; Ascorbic acid; Cd-induced oxidative stress; Glutathione depletion; HO-1 gene expression

Protective effect of ascorbic acid on cadmium-induced hypertension and vascular dysfunction in mice by Wanida Donpunha; Upa Kukongviriyapan; Kwanjit Sompamit; Poungrat Pakdeechote; Veerapol Kukongviriyapan; Patchareewan Pannangpetch (105-115).
Cadmium (Cd) is one of the most important environmental pollutants that cause a number of adverse health effects in humans and animals. Recent studies have shown that Cd-induced oxidative damage within the vascular tissues results in vascular dysfunction. The current study was aimed to investigate whether ascorbic acid could protect against Cd-induced vascular dysfunction in mice. Male ICR mice were received CdCl2 (100 mg/l) via drinking water for 8 weeks alone or received ascorbic acid supplementation at doses of 50 and 100 mg/kg/day for every other day. Results showed that Cd administration increased arterial blood pressure and blunted the vascular responses to vasoactive agents. These alterations were related to increased superoxide production in thoracic aorta, increased urinary nitrate/nitrite, increased plasma protein carbonyl, elevated malondialdehyde (MDA) concentrations in plasma and tissues, decreased blood glutathione (GSH), and increased Cd contents in blood and tissues. Ascorbic acid dose-dependently normalized the blood pressure, improved vascular reactivities to acetylcholine (ACh), phenylephrine (Phe) and sodium nitroprusside (SNP). These improvements were associated with significant suppression of oxidant formation, prevention of GSH depletion, and partial reduction of Cd contents in blood and tissues. The findings in this study provide the first evidence in pharmacological effects of ascorbic acid on alleviation of oxidative damage and improvement of vascular function in a mouse model of Cd-induced hypertension and vascular dysfunction. Moreover, our study suggests that dietary supplementation of ascorbic acid may provide beneficial effects by reversing the oxidative stress and vascular dysfunction in Cd-induced toxicity.
Keywords: Ascorbic acid; Cadmium; Hypertension; Oxidative stress; Vascular dysfunction

Cell cycle arrest in cultured neuroblastoma cells exposed to a bis(thiosemicarbazonato) metal complex by Laura Bica; Jodi Meyerowitz; Sarah J. Parker; Aphrodite Caragounis; Tai Du; Brett M. Paterson; Kevin J. Barnham; Peter J. Crouch; Anthony R. White; Paul S. Donnelly (117-133).
Brain tumors such as neuroblastomas and gliomas are often refractory to current treatments. Development of metal-based drugs may offer an alternative approach due to the ability to deliver radionuclides or cytotoxic metals to the tumor. Previous studies have shown that diacetyl-bis(N(4)-methylthiosemicarbazonato)-copper(II) (CuII(atsm)) can selectively target hypoxic tumors and this feature has been utilized for development of imaging and radiotherapy. However, we have recently shown that glyoxal-bis(N(4)-methylthiosemicarbazonato)-copper(II) (CuII(gtsm)) can target the brain in animal models of neurodegeneration. Unlike CuII(atsm), CuII(gtsm) is able to release Cu intracellularly under normoxic conditions. Glyoxal-bis(thiosemicarbazones) have reported anticancer effects but little is known about the cellular mechanisms involved. Therefore, in this study, we used protein microarray analysis to investigate the effect of CuII(gtsm) on neuroblastoma cell growth in vitro. Treatment of the human neuroblastoma cell line BE(2)-M17, resulted in cell cycle arrest as assessed by fluorescent activated cell sorting (FACS) analysis. Rapidly arrested growth was not associated with onset of apoptosis. Instead, protein microarray analysis revealed that CuII(gtsm) rapidly and potently reduced cyclin D1 expression, while increasing Kip2 expression. Other changes observed were decreased Cdk7 expression and activation of CHK2. These changes may be associated with the cell cycle arrest. We also observed a potent decrease of total and phosphorylated insulin-like growth factor receptor (IGF-IR) by CuII(gtsm) which is associated with modulation of cyclin D1 expression. Our studies reveal important insights into the potential anticancer activity of CuII(gtsm). Further studies are needed to examine the therapeutic potential of CuII(gtsm) and other bis(thiosemicarbazonato) metal complexes as metallo-drugs for treatment of systemic or brain tumors.
Keywords: Copper; Cell cycle; Bis(thiosemicarbazone); Protein microarray

Antibacterial effect of silver nanoparticles on Staphylococcus aureus by Wen-Ru Li; Xiao-Bao Xie; Qing-Shan Shi; Shun-Shan Duan; You-Sheng Ouyang; Yi-Ben Chen (135-141).
The antibacterial activity and mechanism of silver nanoparticles (Ag-NPs) on Staphylococcus aureus ATCC 6538P were investigated in this study. The experiment results showed the minimum bactericidal concentration (MBC) of Ag-NPs to S. aureus was 20 μg/ml. Moreover, when bacteria cells were exposed to 50 μg/ml Ag-NPs for 6 h, the cell DNA was condensed to a tension state and could have lost their replicating abilities. When S. aureus cells were exposed to 50 μg/ml Ag-NPs for 12 h, the cell wall was breakdown, resulting in the release of the cellular contents into the surrounding environments, and finally became collapsed. And Ag-NPs could reduce the enzymatic activity of respiratory chain dehydrogenase. Furthermore, the proteomic analysis showed that the expression abundance of some proteins was changed in the treated bacterial cell with Ag-NPs, formate acetyltransferase increased 5.3-fold in expression abundance, aerobic glycerol-3-phosphate dehydrogenase decreased 6.5-fold, ABC transporter ATP-binding protein decreased 6.2-fold, and recombinase A protein decreased 4.9-fold.
Keywords: Silver nanoparticles (Ag-NPs); Staphylococcus aureus ; Antibacterial effect

Influence of zinc and zinc chelator on HT-29 colorectal cell line by K. S. Gurusamy; N. Farooqui; M. Loizidou; S. Dijk; J. W. Taanman; S. Whiting; M. J. Farquharson; B. J. Fuller; B. R. Davidson (143-151).
Trace elements are involved in many key pathways involving cell cycle control. The influence of zinc and zinc chelator (TPEN) on transcription levels of the main zinc transporters (ZnT1 and ZIP1) in the HT-29 colorectal cell line has not been reported. Proliferation of HT-29 cells was measured using the methylene blue assay after exposure to zinc (two concentrations), TPEN (two concentrations), or a combination of zinc and TPEN (simultaneously and sequentially) for 4 h, 8 h, and 24 h. The transcription levels of ZnT1, ZIP1, vascular endothelial growth factor (VEGF), and caspase-3 were determined using reverse transcriptase real-time polymerase chain reaction (RT-PCR) after exposure of cells to zinc and TPEN. The zinc content in the substrate (medium used for culture) was determined using atomic absorption spectrometry. TPEN decreased cellular proliferation causing complete cell death by 8 h. Zinc had a protective effect against short periods of exposure to TPEN. There was no correlation between the transcripts of main zinc transporters and the zinc content in the substrate. The zinc content in the substrate remained constant after varying periods of cell culture. TPEN decreased the transcript levels of caspase-3 and VEGF, which are surrogate markers for apoptosis and angiogenesis. Zinc chelation of HT-29 cells causes cell death. Zinc appears to be protective for short periods of exposure to TPEN but has no protective effect on prolonged exposure. HT-29 cells are not able to counteract the effect of intracellular chelation of zinc by altering zinc transport. Further research into the mechanisms of these findings is necessary and may lead to novel therapeutic options.
Keywords: Zinc; Zinc chelator; Angiogenesis; Apoptosis; HT-29

Hydroxamate siderophores of the ectomycorrhizal fungi Suillus granulatus and S. luteus by Kurt Haselwandter; Gerlinde Häninger; Markus Ganzera (153-157).
Despite indications that S. granulatus and S. luteus release iron-chelating compounds, the exact spectrum of ferric hydroxamates synthesized by these two Suillus species remained unclear. Hence the aim of this study was to identify all of the main siderophores produced by these two ectomycorrhizal fungal species under pure culture conditions. By means of HPLC and LC–MS analyses we show that S. granulatus releases cyclic and linear fusigen, ferrichrome, coprogen and triacetylfusarinine C into the nutrient medium, while S. luteus culture filtrates contain cyclic and linear fusigen, ferricrocin and coprogen. All of the different siderophores were identified on basis of reference compounds and their specific MS spectra which were recorded on a high resolution MS in positive electrospray ionisation mode. Initial HPLC separations were performed on a C-18 stationary phase, using an acidic eluent (0.1% formic acid in water and acetonitrile) in gradient mode. The potential of these two ectomycorrhizal fungal species to produce siderophores representing three different groups of hydroxamates is discussed in relation to its ecological significance.
Keywords: Mycorrhizal symbiosis; Basidiomycota; Fungal siderophores; Hydroxamates; Iron mobilization

Vesicular distribution of Secretory Pathway Ca2+-ATPase isoform 1 and a role in manganese detoxification in liver-derived polarized cells by Sharon Leitch; Mingye Feng; Sabina Muend; Lelita T. Braiterman; Ann L. Hubbard; Rajini Rao (159-170).
Manganese is a trace element that is an essential co-factor in many enzymes critical to diverse biological pathways. However, excess Mn2+ leads to neurotoxicity, with psychiatric and motor dysfunction resembling parkinsonism. The liver is the main organ for Mn2+ detoxification by excretion into bile. Although many pathways of cellular Mn2+ uptake have been established, efflux mechanisms remain essentially undefined. In this study, we evaluated a potential role in Mn2+ detoxification by the Secretory Pathway Ca2+, Mn2+-ATPase in rat liver and a liver-derived cell model WIF-B that polarizes to distinct bile canalicular and sinusoidal domains in culture. Of two known isoforms, only secretory pathway Ca2+-ATPase isoform 1 (SPCA1) was expressed in liver and WIF-B cells. As previously observed in non-polarized cells, SPCA1 showed overlapping distribution with TGN38, consistent with Golgi/TGN localization. However, a prominent novel localization of SPCA1 to an endosomal population close to, but not on the basolateral membrane was also observed. This was confirmed by fractionation of rat liver homogenates which revealed dual distribution of SPCA1 to the Golgi/TGN and a fraction that included the early endosomal marker, EEA1. We suggest that this novel pool of endosomes may serve to sequester Mn2+ as it enters from the sinusoidal/basolateral domains. Isoform-specific partial knockdown of SPCA1 delayed cell growth and formation of canalicular domain by about 30% and diminished viability upon exposure to Mn2+. Conversely, overexpression of SPCA1 in HEK 293T cells conferred tolerance to Mn2+ toxicity. Taken together, our findings suggest a role for SPCA1 in Mn2+ detoxification in liver.
Keywords: Manganese; Ca2+-ATPase; Liver; Fractionation; Trans-Golgi network; SPCA1; ATP2C1

Cardioprotective effect of zinc requires ErbB2 and Akt during hypoxia/reoxygenation by Kasi Viswanath; Sreedhar Bodiga; Victor Balogun; Anita Zhang; Vijaya Lakshmi Bodiga (171-180).
Recent literature suggests that exogenous zinc can prevent ischemia reperfusion injury by activating phosphoinositide-3 kinase (PI3K)/Akt and by targeting the mitochondrial permeability transition pore (mPTP). It is known that ErbB2 expression promotes association and activation of PI3-kinase/Akt, resulting in growth and survival of cardiac myocytes. In this study, we found that zinc-induced ErbB2 protein expression and Akt activation are required for preventing reperfusion injury. Neonatal rat cardiac myocytes subjected to 8 h of hypoxia, followed by 16 h of reoxygenation induced cardiomyocyte apoptosis, as assessed by increased caspase-3 activity, annexin V staining and lowered MTT reduction and ATP levels. However, addition of zinc-pyrithione (ZPT) before onset of reoxygenation effectively lowered the apoptotic indices and restored the ATP levels. ZPT induced a significant increase in ErbB2 protein expression and Akt activation. Pretreatment with Hsp 90 inhibitor, geldanamycin or PI3-kinase inhibitor, wortmannin prevented the increase in ATP levels and abrogated the protective effect of zinc-pyrithione. Taken together, these data suggest that zinc prevents reperfusion injury by modulating the ErbB2 protein expression and Akt activation.
Keywords: Zinc; Cardiomyocytes; Hypoxia; Reoxygenation; ErbB2; Akt; Hsp90