The effect of prior exercise on ex vivo induction of heme oxygenase-1 in human lymphocytes

It was postulated that prior demanding exercise would suppress the induction of the oxidant-responsive protein Heme Oxygenase-1 (HO-1) in mononuclear cells following subsequent ex vivo H(2)O(2) treatment. Eight male subjects completed two trials in a randomized order (one rest and one exercise) and ex vivo HO-1 protein induction was determined following H(2)O(2) treatment in lymphocytes and monocytes before and after each trial using a newly developed and reproducible assay. Lymphocytes obtained 2 h post-exercise showed a modest reduction in HO-1 protein expression in response to ex vivo treatment with H(2)O(2) (p<0.05). The plasma concentration of the HO-1 suppressor alpha1-antitrypsin increased immediately post-exercise (p<0.05) and it is tentatively suggested that this may explain the modest transient reduction in ex vivo HO-1 protein induction in lymphocytes in response to an independent oxidant challenge following a prior bout of demanding exercise.     

Antioxidation and Antiapoptosis Characteristics of Heme Oxygenase-1 Enhance Tumorigenesis of Human Prostate Carcinoma Cells

Heme oxygenase-1 (HO-1) has antiinflammatory and antioxidant properties and is deemed as a tissue protector. However, effects of HO-1 in prostate cancer remain in controversy. We evaluated the role of HO-1 in prostate carcinoma in vitro and in vivo.Overexpression of HO-1 did not affect prostate cell proliferation in the normal condition but enhanced cell proliferation under serum starvation. HO-1 overexpression enhanced cell invasion of PC-3 cells through epithelial–mesenchymal transition (EMT) induction, which was supported by increased Slug, N-cadherin, and vimentin expressions. In the xenograft animal study, HO-1 overexpression enhanced PC-3 cell tumor growth in vivo. HO-1 attenuated reactive oxygen species induced by H₂O₂ or pyocyanin treatment in PC-3 and DU145 cells. HO-1 further reduced PC-3 and DU145 cell apoptosis induced by H₂O₂ or serum starvation. Our results suggested that HO-1 was able to increase prostate carcinoma cell invasion in vitro and tumor growth in vivo. The EMT induction and antioxidant and antiapoptotic effects of HO-1 in the prostate carcinoma cells may be responsible for these findings.     

The effects of a glycogen loading regimen on acid-base status and blood lactate concentration before and after a fixed period of high intensity exercise in man

Six healthy male subjects exercised after an overnight fast for a fixed 3 min period at a workload equivalent to 100% of their maximal oxygen uptake ( ) on 3 separate occasions. The first test took place after subjects had consumed a mixed diet (43±3% carbohydrate (CHO), 41±5% fat and 16±3% protein) for 3 days, and was followed 2 h later by prolonged cycling to exhaustion at 77±3% to deplete muscle glycogen stores. Following this, subjects consumed a low CHO diet (4±1% CHO, 63±5% fat and 33±6% protein) for the remainder of the day and for the subsequent 2 days; on the morning of the next day a second high intensity test took place. Finally subjects followed a 3 day high CHO diet (73±7% CHO, 17±6% fat and 10±1% protein) before their last test. Acid-base status and selected metabolites were measured on arterialised-venous blood at rest prior to exercise and at intervals for 15 min following exercise. Prior to exercise, plasma pH and blood lactate concentration were higher (p<0.05) after the high CHO diet when compared with the low CHO diet. Pre-exercise plasma bicarbonate, blood PCO₂ and blood base excess were all higher after the high (p<0.001,p<0.01,p<0.01 respectively) and normal (p<0.05,p<0.05,p<0.05 respectively) CHO diets when compared with the low CHO diet. During the post-exercise period there were no differences in plasma pH or blood base excess between the three experimental situations; plasma bicarbonate was higher (p<0.05) at 2 min post-exercise after the high CHO diet when compared with the low CHO diet; blood PCO₂ was higher throughout the post-exercise period after the high CHO diet when compared with the low CHO diet and at 2 min post-exercise was higher after the normal CHO diet than after the low CHO diet (p<0.5). The post-exercise blood lactate concentration after the high CHO diet was at all times higher than the corresponding values recorded after the normal CHO diet and until 15 min post-exercise was significantly higher than the values recorded after the low CHO diet. The present experiment further substantiates the view that a pattern of dietary and exercise manipulation can significantly influence the acid-base status of the blood and by doing so may influence high intensity exercise performance.     

The effects of high-intensity intermittent exercise on the plasma concentrations of glutamine and organic acids

Glutamine is an essential substrate for the proper functioning of cells of the immune system. Falls in plasma glutamine concentration after exercise may have deleterious consequences for immune cell function and render the individual more susceptible to infection. The purpose of the present study was to examine changes in plasma glutamine concentration (measured using a validated enzymatic spectrophotometric method) following an acute bout of intermittent high-intensity exercise. Eight well-trained male games players took part in the study. Subjects reported to the laboratory following an overnight fast and performed a 1-h cycle exercise task consisting of 20 1-min periods at 100% maximal O₂ consumption (V˙O_2max) each separated by 2 min of recovery at 30% V˙O_2max. Venous blood samples were taken before exercise and at 5 min, 1 h, 2.5 h, 5 h and 24 h post-exercise. Glutamine was measured by enzymatic spectrophotometric determination of the ammonia concentration before and after treatment of the plasma with glutaminase (EC 3.5.1.2). Plasma glutamine concentration did not fall in the immediate post-exercise period [pre-exercise 681 (23) μM compared with 663 (46) μM at 5 min post-exercise, mean (SEM)], but fell to 572 (35) μM at 5 h post-exercise (P < 0.05 compared with pre-exercise). Plasma lactate concentration rose to 8.8 (1.0) mM at the end of exercise and fell to 1.8 (0.4) mM at 1 h post-exercise, but plasma concentrations of free fatty acids and β-hydroxybutyrate both rose substantially in the post-exercise period (to 240% and 400% of pre-exercise levels, respectively). The circulating leucocyte count increased significantly during exercise (P < 0.01), continued to increase in the hours following exercise and peaked at 2.5 h post-exercise (mainly due to a neutrophilia). The fall in the plasma glutamine concentration at 5 h post-exercise could be due to increased renal uptake of glutamine, which generally occurs in conditions of metabolic acidosis or due to a greater removal of glutamine from the plasma resulting from the elevated circulating leucocyte count. Accepted: 22 October 1997     

BMP6 attenuates oxidant injury in HK-2 cells via Smad-dependent HO-1 induction

Oxidative stress is involved in a variety of kidney diseases, and heme oxygenase 1 (HO-1) induction is a protective response to oxidative stress. Downregulation of bone morphogenetic protein 6 (BMP6) is associated with renal damage in intrauterine growth-restricted newborns. However, it is unknown whether BMP6 has a renoprotective effect or HO-1 induction property. In this study, we demonstrate that BMP6 effectively protects renal proximal tubule cells (HK-2) against hydrogen peroxide (H₂O₂)-induced cell injury. BMP6 also increased HO-1 gene expression and activity of HO. Inhibition of de novo gene expression, the HO inhibitor ZnPPIX, HO-1 knockdown, or the carbon monoxide (CO) scavenger hemoglobin attenuated the cytoprotective effect of BMP6, whereas HO-1 constitutive expression, the HO-1 inducer hemin, or the hemin metabolites bilirubin and CO ameliorated H₂O₂-induced cell injury. Stimulation of HK-2 cells with BMP6 activated Smad signaling but not mitogen-activated protein kinases. In addition, BMP6-mediated induction of HO-1 expression and increase in HO activity were inhibited by Smad5 knockdown. Furthermore, deletion or mutation of the Smad-binding element in the HO-1 promoter also inhibited BMP6-induced luciferase activity. In summary, these findings suggest that induction of HO-1 through a Smad-dependent mechanism is responsible for the cytoprotective effect of BMP6 in H₂O₂-mediated renal cell injury.     

The Cytoprotective Effects of E-α-(4-Methoxyphenyl)-2’,3,4,4'-Tetramethoxychalcone (E-α-p-OMe-C6H4-TMC)—A Novel and Non-Cytotoxic HO-1 Inducer

Cell protection against different noxious stimuli like oxidative stress or chemical toxins plays a central role in the treatment of many diseases. The inducible heme oxygenase isoform, heme oxygenase-1 (HO-1), is known to protect cells against a variety of harmful conditions including apoptosis. Because a number of medium strong electrophiles from a series of α-X-substituted 2’,3,4,4’-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO₂-C₆H₄, Ph, p-OMe-C₆H₄, NO₂, CF₃, COOEt, COOH) had proven to activate Nrf2 resulting in HO-1 induction and inhibit NF-κB downstream target genes, their protective effect against staurosporine induced apoptosis and reactive oxygen species (ROS) production was investigated. RAW264.7 macrophages treated with 19 different chalcones (15 α-X-TMCs, chalcone, 2’-hydroxychalcone, calythropsin and 2’-hydroxy-3,4,4’-trimethoxychalcone) prior to staurosporine treatment were analyzed for apoptosis and ROS production, as well as HO-1 protein expression and enzyme activity. Additionally, Nrf2 and NF-κB activity was assessed. We found that amongst all tested chalcones only E-α-(4-methoxyphenyl)-2’,3,4,4''-tetramethoxychalcone (E-α-p-OMe-C₆H₄-TMC) demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C₆H₄-TMC displayed no significant activity. Also, E-α-p-OMe-C₆H₄-TMC induced HO-1 protein expression and increased HO-1 activity, whilst inhibition of HO-1 by SnPP-IX abolished its antiapoptotic effect. The only weakly electrophilic chalcone E-α-p-OMe-C₆H₄-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus. Furthermore, staurosporine induced NF-κB activity was attenuated following E-α-p-OMe-C₆H₄-TMC treatment. Overall, E-α-p-OMe-C₆H₄-TMC demonstrated its effective cytoprotective potential via a non-toxic induction of HO-1 in RAW264.7 macrophages. The observed cytoprotective effect may partly be related to both, the activation of the Nrf2- and inhibition of the NF-κB pathway.     

Bacillus amyloliquefaciens SC06 alleviates the oxidative stress of IPEC-1 via modulating Nrf2/Keap1 signaling pathway and decreasing ROS production

Oxidative stress (OS) plays a major role in the gastrointestinal disorders. Although probiotics were reported to repress OS, few researches compared the antioxidant ability of different Bacillus strains and deciphered the mechanisms. To select a Bacillus strain with higher antioxidant capacity, we used H₂O₂ to induce intestinal porcine epithelial cell 1 (IPEC-1) OS model. The most suitable H₂O₂ concentration and incubation time were determined by the half lethal dose and methyl thiazolyl tetrazolium. Correlation analysis was performed to choose a sensitive indicator for OS. As for the comparison of Bacillus, cells were divided into control, Bacillus treatment, H₂O₂ treatment, and Bacillus pre-protection + H₂O₂ treatment. Bacillus were co-cultured with IPEC-1 for 3 h in Bacillus and Bacillus pre-protection + H₂O₂ treatments. Then, based on OS model, 300 μmol/L H₂O₂ was added into medium of H₂O₂ and Bacillus pre-protection + H₂O₂ treatments for another 12 h. Antioxidant and apoptosis gene expressions were detected to screen the target strain. Nuclear factor erythroid-derived 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein1 (Keap1) pathway, reactive oxygen species (ROS) production, mitochondrial membrane potential (Δψm), apoptosis, and necrosis were analyzed. Results revealed that heme oxygenase-1 (HO-1) gene expression had a positive correlation with H₂O₂ induction. Moreover, Bacillus amyloliquefaciens SC06 (SC06)-meditated IPEC-1 showed the best antioxidant capacity though modulating Nrf2 phosphorylation. Δψm was elevated, while ROS generation was reduced with SC06 pre-protection, resulting in decreased apoptosis and necrosis. Altogether, HO-1 expression could be regarded as an OS indicator. The regulation of Nrf2/Keap1 pathway and ROS production by SC06 are involved in alleviating OS of IPEC-1.

     

Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress

Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in cellular defence against oxidative stress by inducing the expression of multiple anti-oxidant genes. However, where high levels of oxidative stress are observed, such as chronic obstructive pulmonary disease (COPD), Nrf2 activity is reduced, although the molecular mechanism for this defect is uncertain. Here, we show that down-regulation of histone deacetylase (HDAC) 2 causes Nrf2 instability, resulting in reduced anti-oxidant gene expression and increase sensitivity to oxidative stress. Although Nrf2 protein was clearly stabilized after hydrogen peroxide (H₂O₂) stimulation in a bronchial epithelial cell line (BEAS2B), Nrf2 stability was decreased and Nrf2 acetylation increased in the presence of an HDAC inhibitor, trichostatin A (TSA). TSA also reduced Nrf2-regulated heme-oxygenase-1 (HO-1) expression in these cells, and this was confirmed in acute cigarette-smoke exposed mice in vivo. HDAC2 knock-down by RNA interference resulted in reduced H₂O₂-induced Nrf2 protein stability and activity in BEAS2B cells, whereas HDAC1 knockdown had no effect. Furthermore, monocyte-derived macrophages obtained from healthy volunteers (non-smokers and smokers) and COPD patients showed a significant correlation between HDAC2 expression and Nrf2 expression (r = 0.92, p < 0.0001). Thus, reduced HDAC2 activity in COPD may account for increased Nrf2 acetylation, reduced Nrf2 stability and impaired anti oxidant defences.     

Fibroblast growth factor-8 inhibits oxidative stress-induced apoptosis in H9c2 cells

Fibroblast growth factors (FGFs) comprise a large family of signaling molecules that involve cell patterning, mobilization, differentiation, and proliferation. Various FGFs, including FGF-1, FGF-2, and FGF-5, have been shown to play a role in cytoprotection during adverse cardiac events; however, whether FGF-8 is a cytoprotective remains unclear. The current study was designed to evaluate the effect of FGF-8 treatment on oxidative stress-induced apoptosis in H9c2 cells. Cells were divided into three groups: control, H₂O₂ (400 µm H₂O₂), and H₂O₂ + FGF-8 (4 ng/ml FGF-8). Our results suggest apoptosis was significantly (p < 0.05) enhanced in the H₂O₂ group relative to control. Moreover, a significant (p < 0.05) decline in apoptosis was observed in the H₂O₂ + FGF-8 group compared to H₂O₂-treated cells as evidenced by TUNEL staining, a cell death detection ELISA, and cell viability. Levels of downstream apoptotic mediators, caspase-3 and caspase-9, were significantly (p < 0.05) upregulated following H₂O₂ treatment but were abrogated following FGF-8 application. Expression levels of Forkhead box protein O1 (FoxO-1), MnSOD, catalase, pAKT, and p-mTOR were significantly (p < 0.05) reduced in the H₂O₂ group (p < 0.05). Notably, these levels were significantly (p < 0.05) reversed following FGF-8 treatment. Our data, for the first time, suggest FGF-8 is an anti-apoptotic mediator in oxidative-stressed H9c2 cells. Furthermore, our data demonstrate that apoptotic inhibition by FGF-8 is consequent to FoxO-1 oxidative detoxification as well as augmentation to the PI3K/AKT cell survival pathway.     

Alterations of erythrocyte structure and cellular susceptibility in patients with chronic renal failure: Effect of haemodialysis and oxidative stress

The aim of this study was to investigate erythrocytes rheological behaviour, membrane dynamics and erythrocytes susceptibility to disintegration upon strong oxidative stress induced by dialysis or by external H₂O₂ among patients with CRF. EPR spectrometry was used to investigate alterations in physical state of cellular components. Generated ROS production induced: (1) significant increase of membrane fluidity in CRF erythrocytes treated with H₂O₂ (p<0.005) and at 60 min of haemodialysis (p<0.05), (2) significant decrease of cytoskeletal protein–protein interactions (p<0.005) and (3) cellular osmotic fragility (p<0.0005). H₂O₂ exacerbated these changes. Erythrocytes from CRF patients have changed rheological behaviour and present higher susceptibility to disintegration. Erythrocytes membrane characteristics indicate that CRF patients possess younger and more flexible cells, which are more susceptible to oxidative stress. This may contribute to the shortened survival of young erythrocytes in CRF patients.     

An aqueous extract of Zingiber officinale Roscoe protects mouse primary hepatic cells against hydrogen peroxide-induced oxidative stress

Hepatocytes exposed to an oxidative stressor such as hydrogen peroxide (H₂O₂) are potentially sensitized to cell death; thus, reactive oxygen species (ROS) are considered to be critical mediators of liver damage. Zingiber officinale Roscoe (ZO), also known as ginger, is cultivated commercially in China, India, Korea, and other parts of the world. In addition, it is used as a spice and flavoring agent and is also purported to possess a number of medicinal properties. In the present study, we examined the protective effect of ZO against cell damage caused by H₂O₂-induced oxidative stress. ZO reduced H₂O₂-induced apoptotic signals and the levels of intracellular ROS. ZO pretreatment also increased the phosphorylation of c-Jun, and JNK kinase. The expression of heme oxygenase-1 (HO-1) and heat shock protein 72 (HSP72) were increased by ZO pretreatment more than H₂O₂ treatment. In addition, siRNA-mediated knockdown of HO-1 and HSP72 decreased protective effect of ZO pretreatment. Our data suggest that ZO decreases ROS levels and the expressions of HO-1 and HSP72 are involved in the hepatocyte protective function of ZO against H₂O₂.     

GDNF modulates HO-1 expression in substantia nigra postnatal cell cultures

Heme oxygenase-1 (HO-1) has been strongly highlighted because of its induction in many cell types by toxic stimuli, including oxidative stress. The intense HO-1 immunostaining in the substantia nigra of Parkinson disease (PD) patients suggests its involvement in the pathogenesis of this neurodegenerative disease. In this work we investigated HO-1 expression in rat substantia nigra postnatal cell cultures under conditions mimicking dopamine toxicity and its modulation by glial cell line-derived neurotrophic factor (GDNF), a potent neuroprotective factor for dopaminergic neurons. In neuron–glia cultures, we found that H₂O₂, a product of dopamine metabolism, or l-3,4-dihydroxyphenylalanine (l-DOPA), the dopamine precursor used in the therapy of PD, induced a fast up-regulation of HO-1 mRNA and protein levels, followed by a secondary down-regulation. H₂O₂ and l-DOPA also increased HO-1 expression in astrocyte cultures, but with a delayed time course in H₂O₂-treated cultures. HO-1 expression was decreased in neuron–glia cultures under conditions under which GDNF up-regulation was observed. Because exogenously applied GDNF prevented HO-1 up-regulation in cultures treated with H₂O₂ or l-DOPA, and antibody neutralization of GDNF prevented the secondary HO-1 down-regulation observed in neuron–glia cultures, we propose that GDNF negatively modulates HO-1 expression induced by oxidative stress. To our knowledge, this is the first report showing the modulation of HO-1 expression by GDNF.     

Effect of meal size on excess post-exercise oxygen consumption in fishes with different locomotive and digestive performance

Effects of feeding on pre-exercise VO₂ and excess post-exercise oxygen consumption (EPOC) after exhaustive exercise were investigated in sedentary southern catfish, active herbivorous grass carp, omnivorous crucian carp, and sluggish omnivorous darkbarbel catfish to test whether feeding had different effects on EPOC and to compare EPOC in fishes with different ecological habits. For fasting fish, the pre-exercise and peak post-exercise VO₂ were higher and recovery rates were faster in crucian carp and grass carp compared to those of darkbarbel catfish and southern catfish. EPOC magnitudes of grass carp and southern catfish were significantly larger than those of crucian carp and darkbarbel catfish. Feeding had no significant effect on peak post-exercise VO₂, recovery rate, and EPOC magnitude in grass carp. Both the pre-exercise and peak post-exercise VO₂ increased with meal size, while the EPOC magnitude and duration decreased significantly in the larger meal size groups of crucian carp and southern catfish. In darkbarbel catfish, both the pre-exercise and peak post-exercise VO₂ increased with meal size, but the VO₂ increment elicited by exercise was larger in feeding groups compared with the fasting group. These results suggest that (1) the characteristics of the post-exercise VO₂ profile, such as peak post-exercise VO₂ and recovery rate, were closely related to the activity of fishes, whereas the EPOC magnitude was not and (2) the effects of feeding on EPOC were more closely related to the postprandial increase in VO₂.     

Ataxia telangiectasia mutated inhibits oxidative stress-induced apoptosis by regulating heme oxygenase-1 expression

Ataxia telangiectasia (AT) is caused by mutational inactivation of the ataxia telangiectasia mutated (Atm) gene, which is involved in DNA repair. Increased oxidative stress has been shown in human AT cells and neuronal tissues of Atm-deficient mice. Heme oxygenase-1 (HO-1) is an inducible antioxidant enzyme and protects cells against oxidative stress. The purpose of this study is to determine whether ATM induces antioxidant enzyme HO-1 and protects cells from oxidative stress-mediated apoptosis by driving the activation of PKC-δ and NF-κB, by increasing cell viability, and by downregulating DNA fragmentation and apoptotic indicators (apoptosis-inducing factor and cleaved caspase-3). AT fibroblasts stably transfected with human full-length ATM cDNA (YZ5 cells) or the empty vector (MOCK cells) were treated with H₂O₂ as a source of reactive oxygen species (ROS). As a result, transfection with ATM inhibited ROS-induced cell death and DNA fragmentation in MOCK cells. Transfection with ATM induced expression of HO-1 which was mediated by PKC-δ and NF-κB in H₂O₂-treated MOCK cells. ZnPP, an HO-1 inhibitor, and transfection with HO-1 siRNA increased ROS levels and apoptosis, whereas hemin, an HO-1 activator, reduced ROS levels and apoptosis in H₂O₂-treated YZ5 cells. Rottlerin, a PKC-δ inhibitor, inhibited NF-κB activation and HO-1 expression in H₂O₂-treated YZ5 cells. MOCK cells showed increased cell death, DNA fragmentation, and apoptotic indicators compared to YZ5 cells exposed to H₂O₂. In addition, transfection with p65 siRNA increased ROS levels and DNA fragmentation, but decreased HO-1 protein levels in H₂O₂-treated YZ5 cells. In conclusion, ATM induces HO-1 expression via activation of PKC-δ and NF-κB and inhibits oxidative stress-induced apoptosis. A loss of HO-1 induction may explain why AT patients are vulnerable to oxidative stress.     

Cocoa polyphenols attenuate hydrogen peroxide-induced inhibition of gap-junction intercellular communication by blocking phosphorylation of connexin 43 via the MEK/ERK signaling pathway

Cocoa, a good source of dietary antioxidative polyphenols, exhibited anticarcinogenic activity in animal models, but the molecular mechanisms of the chemopreventive potential of cocoa remain unclear. Inhibition of gap-junction intercellular communication (GJIC) is strongly related to tumorigenesis. Cocoa polyphenol extracts (CPE) dose dependently attenuated hydrogen peroxide (H₂O₂)-induced inhibition of GJIC in rat liver epithelial (RLE) cells. CPE inhibited the H₂O₂-induced phosphorylation and internalization of connexin 43, which is a regulating protein of GJIC in RLE cells. The H₂O₂-induced accumulation of reactive oxygen species and activation of extracellular signal-regulated kinase were inhibited by CPE treatment. However, CPE did not block H₂O₂-induced phosphorylation of p38 mitogen-activated protein kinase. An ex vivo kinase assay demonstrated that CPE inhibited the H₂O₂-induced mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 1 activity in RLE cell lysates. Ex vivo pull-down assay data revealed that CPE directly bound with MEK1 to inhibit MEK1 activity. These results indicate that CPE protects against the H₂O₂-induced inhibition of GJIC through antioxidant activity and direct inhibition of MEK activity, which may contribute to its chemopreventive potential.     

Induction of heme oxygenase-1 improves cold preservation effect of liver graft

We have examined the protective effect and mechanisms of heme oxygenase-1 (HO-1) induction in rat liver model of ex vivo cold ischemia preservation using cobalt protoporphyrin (CoPP) as HO-1 inducer and zinc protoporphyrin (ZnPP) as HO-1 inhibitor. There was a decrease in both aspartate transaminase and lactate dehydrogenase activities and in malondialdehyde level in liver of the CoPP-treated group compared with controls (p < 0.05). In the CoPP-treated rats, the histological signs of reperfusion injury were much lower than in control. Up-regulation of HO-1 expression was also associated with reduced levels of tumor necrosis factor α and interleukin-6. Markedly fewer apoptotic liver cells (determined by TUNEL assay) could be detected in CoPP-treated group compared with the control group. These protective effects were prevented by administration of ZnPP. In conclusion, induction of HO-1 provides protection against liver injury during cold ischemia preservation and improves the preservation of liver graft. The mechanisms underlying these beneficial effects include reduction of oxidative injury and of inflammatory response and prevention of apoptosis. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 5, pp. 674–681.     

Salicylic acid and H2O2 function by independent pathways in the induction of freezing tolerance in potato

The effects of salicylic acid (SA) and hydrogen peroxide (H₂O₂) on freezing tolerance were studied in two potato (Solanum tuberosum) cultivars (Alpha and Atlantic) that differ in cold sensitivity, Alpha being more tolerant to freezing than Atlantic. Lowest freezing survival rates were observed in 4-week-old plants. Freezing treatments consisting of exposure to 6° C for 4 h in the dark were applied 24 h after plants had been transferred from in vitro culture to soil. Catalase activity and H₂O₂ were estimated at the following harvest points: stage (a) 4-week-old in vitro plants treated with either 0.1 mM SA or 5 mM H₂O₂; stage (b) as in (a) but 24 h following transfer to soil prior to freezing treatment; stage (c) as in (b) but measured 15 days after a 4-h freezing treatment. The results show that (1) SA induced freezing tolerance in both cultivars; (2) SA inhibited ascorbate peroxidase activities in both cultivars at all harvest points but inhibited catalase activities in only at stage (a); (3) SA induced H₂O₂ accumulation only in Atlantic at stage (a); (4) H₂O₂ enhanced shoot catalase activities in Atlantic at stages (a) and (b) whereas this treatment had no effect on shoot catalase activities in Alpha; (5) H₂O₂ treatment induced freezing tolerance in Atlantic, even though shoot catalase activities were lower than those of the controls following exposure to freezing temperatures. We conclude that SA does not always lead to H₂O₂ accumulation even though catalase and ascorbate peroxidase activities are decreased as a result of the treatment. Moreover, H₂O₂ accumulation is not always associated with the induction of freezing tolerance, for example at stage (a) where SA-induced tolerance in Alpha was not accompanied by H₂O₂ accumulation. H₂O₂ was able to induce freezing tolerance only in Atlantic, even though H₂O₂ accumulated in both cultivars following this treatment.     

Hydrogen peroxide (H<Subscript>2</Subscript>O<Subscript>2</Subscript>) and methyl-β-cyclodextrin (MβCD) down regulate caveolin expression in human lens epithelial cells (HLECs)

Oxidative stimulation induced by hydrogen peroxide (H₂O₂) on human lens epithelial cells (HLECs) was performed to observe the effects on cell viability and caveolin expression, and cholesterol depletion in HLECs caused by methyl-β-cyclodextrin (MβCD) was also studied. SRA01/04 HLECs were exposed to H₂O₂ or MβDC of various concentrations and durations. We used a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to measure the effect of H₂O₂ on the viability of SRA01/04 HLECs. The distributions of caveolins after oxidative stimulation were probed by fluorescence microscopy and laser scanning confocal microscopy. Immunoblotting was performed to analyze alterations of caveolins expression. We observed that the viability of SRA01/04 HLECs under 0.1 mM H₂O₂ for 10 min or longer was significantly reduced (*p < 0.05, F = 11.63). Laser scanning microscopy showed immunofluorescent caveolins in SRA01/04 HLECs under 0.1 mM H₂O₂ for 10 min or longer; caveolins were largely confined to intracellular domains. Western blots showing both membrane and total caveolin protein (22 kDa) levels in SRA01/04 HLECs treated with 0.1, 0.2, 0.5, or 1.0 mM H₂O₂ for 30 min were significantly reduced, compared with the untreated (*p < 0.05, F = 6.149, or *p < 0.05, F = 14.489, respectively). In addition, the membrane and total caveolin protein level after being treated with 0.1 mM H₂O₂ (*p < 0.05, F = 6.843, or *p < 0.05, F = 7.944, respectively) for different durations also down regulated. Fluorescence microscopy also showed that phosphorylated caveolin-1 was distributed near the focal adhesions of the cells. This study concludes that the responses of HLECs to oxidative stress may include down regulation of caveolin and phosphorylation of caveolin-1 on tyrosine 14 and that MβCD also down regulates caveolin while depleting cholesterol in HLECs. Published in Russian in Molekulyarnaya Biologiya, 2007, Vol. 41, No. 6, pp. 994–1001. The article was submitted by the authors in English.