Greater Reactive Oxygen Species Production Research Articles

Diazoxon exposure increases susceptibility to infection by Salmonella Typhimurium

ABSTRACT Various exogenous factors, such as microbiological and chemical contamination condition food security. Salmonella Typhimurium (S. Typhimurium) is the cause of salmonellosis. This bacterium utilizes phagocytosis to create bacterial reservoirs. On the other hand, exposure to chemical contaminants, such as pesticides, increases susceptibility to numerous infections. Therefore, this research aims to evaluate the effect of co-exposure to diazoxon and S. Typhimurium on the in vitro infection dynamics. For this purpose, human mononuclear cells were pre-exposed in vitro to diazoxon and then challenged with S. Typhimurium at 1, 8, and 24 h. Bacterial internalization, actin polymerization, and reactive oxygen species (ROS) were analyzed. Obtained data show that mononuclear cells previously exposed to diazoxon exhibit greater internalization of S. Typhimurium. Likewise, greater ROS production and an increase in actin polymerization were observed. Therefore, in the proposed scenario, obtained data suggest that co-exposure to diazoxon and S. Typhimurium increases susceptibility to acquiring an illness.

Valproic acid inhibits cell growth in both MCF-7 and MDA-MB231 cells by triggering different responses in a cell type-specific manner

BackgroundBreast cancer is the second leading cause of death among women after lung cancer. Despite the improvement in prevention and in therapy, breast cancer still remains a threat, both for pre- and postmenopausal women, due to the development of drug resistance. To counteract that, novel agents regulating gene expression have been studied in both hematologic and solid tumors. The Histone Deacetylase (HDAC) inhibitor Valproic Acid (VA), used for epilepsy and other neuropsychiatric diseases, has been demonstrated a strong antitumoral and cytostatic activity. In this study, we tested the effects of Valproic Acid on the signaling pathways involved in breast cancer cells viability, apoptosis and in Reactive Oxygen Species (ROS) production using ER-α positive MCF-7 and triple negative MDA-MB-231 cells.MethodsCell proliferation assay was performed by MTT Cell cycle, ROS levels and apoptosis were analyzed by flow cytometry, protein levels were detected by Western Blotting.ResultsCell treatment with Valproic Acid reduced cell proliferation and induced G0/G1 cell cycle arrest in MCF-7 and G2/M block in MDA-MB-231 cells. In addition, in both cells the drug enhanced the generation of ROS by the mitochondria. In MCF-7 treated cells, it has been observed a reduction in mitochondrial membrane potential, a down regulation of the anti-apoptotic marker Bcl-2 and an increase of Bax and Bad, leading to release of cytochrome C and PARP cleavage. Less consistent effects are recorded in MDA-MB-231 cells, in which the greater production of ROS, compared to MCF-7cells, involves an inflammatory response (activation of p-STAT3, increased levels of COX2).ConclusionsOur results have demonstrated that in MCF-7 cells the Valproic Acid is a suitable drug to arrest cell growth, to address apoptosis and mitochondrial perturbations, all factors that are important in determining cell fate and health. In a triple negative MDA-MB 231 cells, valproate directs the cells towards the inflammatory response with a sustained expression of antioxidant enzymes. Overall, the not always unequivocal data between the two cellular phenotypes indicate that further studies are needed to better define the use of the drug, also in combination with other chemotherapy, in the treatment of breast tumors.

Mitochondrial aberrations during the progression of disuse atrophy differentially affect male and female mice.

BackgroundDisuse decreases muscle size and is predictive of mortality across multiple pathologies. Detriments to mitochondrial function are hypothesized to underlie disuse‐induced muscle atrophy. Little data exist on early mechanisms contributing to onset of these pathologies, nor is it known how they differ between sexes. The purpose of this study was to examine differential and conserved responses to mitochondrial quality control in male and female mice during the development and progression of disuse‐induced atrophy.MethodsOne hundred C57BL/6J mice (50 male and 50 female) were hindlimb unloaded to induce disuse atrophy for 0 (con), 24, 48, 72, or 168 h. At designated time‐points, extensor digitorum longus, gastrocnemius, and soleus muscles were collected for analysis of mitochondrial quality control markers.ResultsOne hundred sixty‐eight hours of disuse resulted in ~25% lower oxidative muscle fibre CSA in both male (P = 0.003) and female (P = 0.02) mice without any differences due to disuse in glycolytic fibres. In male mice, 48 h of unloading was sufficient to result in ~67% greater mitochondrial oxidative stress as assessed by the reporter gene pMitoTimer compared with 0 h (P = 0.002), this mitochondrial stress preceded detectable muscle loss. However in female mice, mitochondrial oxidative stress did not occur until 168 h of disuse (~40% greater mitochondrial oxidative stress in 168 h compared with 0 h of disuse, P < 0.0001). Blunted oxidative stress in female mice appeared to coincide with greater inductions of autophagy and mitophagy in female mice (~3‐fold greater BNIP3 and ~6‐fold greater LC3II/I ratio P < 0.0001 and P = 0.038 respectively). Male mice overall had greater reactive oxygen species (ROS) production compared with female mice. Female mice had a greater induction of ROS within 24 h of disuse (~4‐fold greater compared with 0 h, P < 0.0001); whereas male mice did not have greater ROS production until 168 h of disuse (~2‐fold greater, P < 0.0001). Although all muscle types exhibited some alterations to mitochondrial quality control, such as increased markers of mitophagy and fission, the soleus muscle in both male and female mice exhibited consistent alterations to various markers of mitochondrial quality. Markers of mitochondrial translation were approximately 30–50% lower within 24 h of unloading in both male and female soleus muscle (P value ranges: <0.0001–0.03).ConclusionsDisuse negatively affects mitochondria differentially between sexes during development of muscle wasting. Acutely, female mice may forgo muscle mass to maintain mitochondrial quality compared with male mice. These differences may contribute to divergent clinical manifestations of atrophy.

Lifelong Ulk1-Mediated Autophagy Deficiency in Muscle Induces Mitochondrial Dysfunction and Contractile Weakness

The accumulation of damaged mitochondria due to insufficient autophagy has been implicated in the pathophysiology of skeletal muscle aging. Ulk1 is an autophagy-related kinase that initiates autophagosome assembly and may also play a role in autophagosome degradation (i.e., autophagy flux), but the contribution of Ulk1 to healthy muscle aging is unclear. Therefore, the purpose of this study was to investigate the role of Ulk1-mediated autophagy in skeletal muscle aging. At age 22 months (80% survival rate), muscle contractile and metabolic function were assessed using electrophysiology in muscle-specific Ulk1 knockout mice (MKO) and their littermate controls (LM). Specific peak-isometric torque of the ankle dorsiflexors (normalized by tibialis anterior muscle cross-sectional area) and specific force of the fast-twitch extensor digitorum longus muscles was reduced in MKO mice compared to LM mice (p < 0.03). Permeabilized muscle fibers from MKO mice had greater mitochondrial content, yet lower mitochondrial oxygen consumption and greater reactive oxygen species production compared to fibers from LM mice (p ≤ 0.04). Alterations in neuromuscular junction innervation patterns as well as changes to autophagosome assembly and flux were explored as possible contributors to the pathological features in Ulk1 deficiency. Of primary interest, we found that Ulk1 phosphorylation (activation) to total Ulk1 protein content was reduced in older muscles compared to young muscles from both human and mouse, which may contribute to decreased autophagy flux and an accumulation of dysfunctional mitochondria. Results from this study support the role of Ulk1-mediated autophagy in aging skeletal muscle, reflecting Ulk1′s dual role in maintaining mitochondrial integrity through autophagosome assembly and degradation.

Effect of Elaeagnus umbellata (Thunb.) fruit extract on H2O2-induced oxidative and inflammatory responses in normal fibroblast cells.

BackgroundElaeagnus umbellata is a plant commonly used in traditional Asian medicine for its many health benefits and strong antioxidative activity. Its therapeutic potential is believed to be connected to its effect on fibroblasts. This study aimed to investigate E. umbellata methanol-acetone extract’s (EUE) defense against hydrogen peroxide (H2O2)-induced fibroblast damage.MethodsBecause the main biologically active compounds of E. umbellata are water-insoluble, we evaluated the effects of methanol-acetone fruit extracts using liquid chromatography (for ascorbic acid and beta-carotene) and spectrophotometry (for lycopene and total phenolics). The extract’s antioxidative activity was measured using DPPH radical inhibition, and EUE’s effect on human fibroblasts was also evaluated. We assessed the metabolic activity and apoptosis of HFFF-2 fibroblasts exposed to EUE and/or H2O2using the XTT test and flow cytometry, respectively. Superoxide dismutase activity and reactive oxygen species (ROS) production were evaluated using colorimetric and fluorometric assays, respectively. We measured pro-inflammatory cytokine (MIF, fractalkine, MCP-4, BLC, GCP-2, NAP-2, Eotaxin-2, and Eotaxin-3) expression in HFFF-2 cells using immunocytochemistry.ResultThe extract increased HFFF-2 cell proliferation and reduced cell death caused by H2O2-induced stress. H2O2-treated fibroblasts had greater ROS production than cells treated with both H2O2 and EUE. Additionally, the group treated with H2O2 alone showed higher pro-inflammatory cytokine (MIF, MCP-4, NAP-2, Eotaxin-2, and Eotaxin-3) expression.ConclusionEUE protected human fibroblasts from H2O2-induced oxidative stress and reduced the fibroblast-mediated inflammatory response triggered by ROS.

Oxidative stress biomarkers in newborn calves: Comparison among artificial insemination, in vitro fertilization and cloning

Oxidative stress occurs when there is greater than optimal production of reactive oxygen species (ROS) or an antioxidant system failure. Calves produced using in vitro fertilization (IVF) or cloning (CA) have greater mortality rates, with greater incidence of respiratory diseases, which could be explained by the deleterious outcomes from oxidative stress. Calves were studied that were produced using: artificial insemination (AI; n = 20), in vitro fertilization (IVF; n = 15) or cloning (CA; n = 15). Blood samples were collected at 6, 12, 24 and 48 h subsequent to the time of birth. The cloned calves had greater ROS production from lipid peroxidation, with greater thiobarbituric acid reactive substances. This factor was associated with a lesser amount of superoxide dismutase in the CA. Calves produced using IVF had a greater activity of catalase and glutathione peroxidase, either due to greater production of hydrogen peroxide or greater efficiency of enzymatic response of these neonates. Calves produced using AI had greater concentrations ​​of reduced thiol groups. These associated factors may indicate there is greater oxidative stress in calves produced by IVF and cloning than with use of AI, however in these calves there was an effective response to these oxidative stressors within 48 h subsequent to birth. Hence, calves produced using IVF and by cloning have greater ROS production when compared to calves produced using AI. The calves produced using IVF, however, had a greater enzymatic activity or were more efficient in adapting to ROS when compared to calves produced by cloning.

Malachite green-conjugated multi-walled carbon nanotubes potentiate antimicrobial photodynamic inactivation of planktonic cells and biofilms of Pseudomonas aeruginosa and Staphylococcus aureus.

Purpose: Infections associated with medical devices that are caused by biofilms remain a considerable challenge for health care systems owing to their multidrug resistance patterns. Biofilms of Pseudomonas aeruginosa and Staphylococcus aureus can result in life-threatening situations which are tough to eliminate by traditional methods. Antimicrobial photodynamic inactivation (aPDT) constitutes an alternative method of killing deadly pathogens and their biofilms using reactive oxygen species (ROS). This study investigated the efficacy of enhanced in vitro aPDT of P. aeruginosa and S. aureus using malachite green conjugated to carboxyl-functionalized multi-walled carbon nanotubes (MGCNT). Both the planktonic cells and biofilms of test bacteria were demonstrated to be susceptible to the MGCNT conjugate. These MGCNT conjugates may thus be employed as a facile strategy for designing antibacterial and anti-biofilm coatings to prevent the infections associated with medical devices.Methods: Conjugation of the cationic dye malachite green to carbon nanotube was studied by UV-visible spectroscopy, high-resolution transmission electron microscopy, and Fourier transform infrared spectrometry. P. aeruginosa and S. aureus photodestruction were studied using MGCNT conjugate irradiated for 3 mins with a red laser of wavelength 660 nm and radiant exposure of 58.49 J cm−2.Results: Upon MGCNT treatment, S. aureus and P. aeruginosa were reduced by 5.16 and 5.55 log10 , respectively. Compared to free dye, treatment with MGCNT afforded improved phototoxicity against test bacteria, concomitant with greater ROS production. The results revealed improved biofilm inhibition, exopolysaccharide inhibition, and reduced cell viability in test bacteria treated with MGCNT conjugate. P. aeruginosa and S. aureus biofilms were considerably reduced to 60.20±2.48% and 67.59±3.53%, respectively. Enhanced relative MGCNT phototoxicity in test bacteria was confirmed using confocal laser scanning microscopy.Conclusion: The findings indicated that MGCNT conjugate could be useful to eliminate the biofilms formed on medical devices by S. aureus and P. aeruginosa.

Old age increases microglial senescence, exacerbates secondary neuroinflammation, and worsens neurological outcomes after acute traumatic brain injury in mice

After traumatic brain injury (TBI), individuals aged over 65 years show increased mortality and worse functional outcomes compared with younger persons. As neuroinflammation is a key pathobiological mechanism of secondary injury after TBI, we examined how aging affects post-traumatic microglial responses and functional outcomes. Young (3-month-old) and aged (18-month-old) male C57Bl/6 mice were subjected to moderate-level controlled cortical impact or sham surgery, and neurological function was evaluated. At 72 hours after injury, brain, blood, and spleen leukocyte counts were assessed ex vivo using flow cytometry. Aged mice demonstrated more severe deficits in forelimb grip strength, balance and motor coordination, spontaneous locomotor activity, and anxiety-like behavior. These animals also exhibited more robust microglial proliferation and significantly higher numbers of brain-infiltrating leukocytes. Microglia in aged mice showed impairments in phagocytic activity and higher production of interleukin-1β (IL-1β). Infiltrating myeloid cells in aged TBI mice also had deficits in phagocytosis but showed diminished proinflammatory cytokine production and greater reactive oxygen species production. Expression of several senescence markers (Bcl-2, p16ink4a, p21cip1a, lipofuscin, and H2AX [pS139]) was increased with age and/or TBI in both microglia and injured cortex. Although there was no difference in the number of circulating blood neutrophils as a function of age, young mice exhibited more pronounced TBI-induced splenomegaly and splenic myeloid cell expansion. Thus, worse post-traumatic behavioral outcomes in aged animals are associated with exaggerated microglial responses, increased leukocyte invasion, and upregulation of senescence markers.

Phytoremediation potential of Salvinia molesta for arsenite contaminated water: role of antioxidant enzymes

Antioxidant enzymes are important components in the defense against arsenic (As) stress in plants. Here, we tested the hypothesis that Salvinia molesta, an aquatic fern, counteracts the harmful arsenite (AsIII) effects by activating scavenging reactive oxygen species (ROS) enzymes. Thus, our objective was to investigate the role of the superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX) in S. molesta tolerance to AsIII and indicate the use of this plant in remediation of contaminated water. Plants were grown in nutrient solution at pH 6.5 and exposed to 0, 5, 10, or 20 µM AsIII for 96 h (analyses of As absorption, mineral nutrient content, and relative growth rate) and for 24 h (analyses of oxidative stress indicators and enzymatic antioxidant defenses). In the floating leaves, there was a greater basal activity of the antioxidant enzymes and less accumulation of As than in submerged leaves. The submerged leaves, which function as roots in S. molesta, accumulated more As than floating leaves, and SOD and CAT activities were inhibited. Thus, there was a greater production of ROS and oxidative stress. Our results show that S. molesta presents enzymatic antioxidant defenses to alleviate AsIII toxicity and are more effectives in the floating leaves. These results are important to elucidate the AsIII tolerance mechanisms in S. molesta and the possibility of their use in contamined water phytoremediation. Additional studies exposing plants to more prolonged stress and using AsIII concentrations closer to those found in contaminated environments will confirm this claim.

Metabolic adaptation of pigs to a Mycoplasma hyopneumoniae and Lawsonia intracellularis dual challenge.

Respiratory and enteric pathogens such as Mycoplasma hyopneumoniae (Mh) and Lawsonia intracellularis (LI) reduce lean accretion and feed efficiency (FE) in growing pigs. However, the metabolic mechanism by which this occurs is still unknown. Therefore, the primary aim of this study was to examine the metabolic adaptation of pigs presented with a dual Mh and LI challenge (MhLI). A secondary objective was to examine if selection for high FE, modeled by selection for low residual feed intake (RFI), alters molecular response to disease. Using a 2 × 2 factorial design, 6 littermate pairs from a high RFI (HRFI) and 6 littermate pairs from a low RFI (LRFI) line (barrows, 66 ± 2 kg BW) were selected, with 1 pig from each pair assigned to individual pens in either the challenge or the nonchallenge (control) rooms (n = 6 barrows per line/challenge). On days post inoculation (dpi) 0, MhLI pigs were inoculated intragastrically with LI and intratracheally with Mh. Pig and feeder weights were recorded at dpi 0, 7, 14, and 21. On dpi 21, pigs were euthanized and tissues and blood were collected. Markers of oxidative stress, skeletal muscle metabolism and proteolysis, and liver gluconeogenesis were evaluated to determine the effects of MhLI, RFI line, and their interaction. The interaction of line and challenge was not significant (P > 0.05) for any measure. Overall, MhLI pigs had lower ADG (38%, P < 0.001), ADFI (25%, P < 0.001), and G:F (19%, P = 0.012) compared with controls. As expected, LRFI pigs had lower ADFI (P = 0.028) for the same ADG, giving them greater G:F (P = 0.021) than HRFI pigs. Challenged pigs had greater reactive oxygen species (ROS) production in the LM and liver (P < 0.10) but did not have greater skeletal muscle proteolysis. Liver gluconeogenesis was also not upregulated (P > 0.05) due to MhLI. These results provide further evidence that selection for LRFI does not negatively affect response to disease. In addition, these results suggest that postabsorptive metabolic functions are altered due to MhLI challenge. The MhLI challenge induced mitochondrial dysfunction, evident by greater ROS production, and caused pigs to favor glycolytic energy generation. However, skeletal muscle proteolysis and liver gluconeogenesis were not upregulated during MhLI challenge. These data suggest that during mild disease stress, pigs can meet energy demands without reliance on nutrient mobilization and gluconeogenesis.

Comparative analysis of innate immune responses to Streptococcus phocae strains in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss).

Streptococcus phocae subsp. salmonis is a Gram-positive bacterium that causes mortality only in Atlantic salmon (Salmo salar) farmed in Chile, even when this species is co-cultured with rainbow trout (Oncorhynchus mykiss). This susceptibility could be determined by innate immune response components and their responses to bacterial infection. This fish pathogen shares subspecies status with Streptococcus phocae subsp. phocae isolated from seals. The present study compared innate immune system mechanisms in Atlantic salmon and rainbow trout when challenged with different S.phocae, including two isolates from Atlantic salmon (LM-08-Sp and LM-13-Sp) and two from seal (ATCC 51973(T) and P23). Streptococcus phocae growth was evaluated in the mucus and serum of both species, with rainbow trout samples evidencing inhibitory effects. Lysozyme activity supported this observation, with significantly higher (p<0.01) expression in rainbow trout serum and mucus as compared to Atlantic salmon. No differences were found in phagocytic capacity between fish species when stimulated with ATCC 51973(T) and P23. Against all S.phocae strains, rainbow trout and Atlantic salmon showed up to two-fold increased bactericidal activity, and rainbow trout demonstrated up to three-fold greater reactive oxygen species production in macrophages. In conclusion, the non-specific humoral and cellular barriers of Atlantic salmon were immunologically insufficient against S.phocae subsp. salmonis, thereby facilitating streptococcosis. Moreover, the more robust response of rainbow trout to S.phocae could not be attributed to any specific component of the innate immune system, but was rather the consequence of a combined response by the evaluated components.

Reactive oxygen species and antioxidant levels as the factors of autumn senescence in phenological forms of beech (Fagus sylvatica L.)

This study examines changes in the reactive oxygen species (ROS) content and in the level and degree of ascorbate and glutathione oxidation during leaf senescence in phenological forms of beech (Fagus sylvatica L.). Leaves were collected between August and October in 2009 and 2012. Chlorophyll degradation depended significantly on the phenological characteristics of the specimens. Chlorophyll, proteins, superoxide ion and hydrogen peroxide contents and lipid peroxidation dynamics exhibited greater dependence on temperature changes in the early form than in the late form. In the early stage of senescence, the late beech form presented a greater increase in ascorbate and glutathione synthesis than did the early form (ascorbate and glutathione synthesis was the main characteristic of the phenological forms’ defence mechanism against increasing ROS concentration). The early form was characterized by a stronger correlation between increasing ROS and the concentrations of oxidized forms of ascorbate and glutathione. We conclude that the earlier onset and greater rate of autumn senescence in the early phenological form relative to the late form were associated with significantly quicker chlorophyll degradation and greater production of ROS, which was better balanced by the antioxidant system in the late phenological form. The different concentrations of ascorbate and glutathione and the degree of their oxidation influenced the oxidative stress tolerance in the senescing leaves of the different phenological forms of beech.

Effect of moderate exercise on peritoneal neutrophils from juvenile rats.

Previous studies showed that moderate exercise in adult rats enhances neutrophil function, although no studies were performed in juvenile rats. We evaluated the effects of moderate exercise on the neutrophil function in juvenile rats. Viability and neutrophils function were evaluated. Moderate exercise did not impair the viability and mitochondrial transmembrane potential of neutrophils, whereas there was greater reactive oxygen species production (164%; p < 0.001) and phagocytic capacity (29%; p < 0.05). Our results suggest that moderate exercise in juvenile rats improves neutrophil function, similar to adults.

The role of mitochondrial DNA mutation on neurodegenerative diseases.

Many researchers have reported that oxidative damage to mitochondrial DNA (mtDNA) is increased in several age-related disorders. Damage to mitochondrial constituents and mtDNA can generate additional mitochondrial dysfunction that may result in greater reactive oxygen species production, triggering a circular chain of events. However, the mechanisms underlying this vicious cycle have yet to be fully investigated. In this review, we summarize the relationship of oxidative stress-induced mitochondrial dysfunction with mtDNA mutation in neurodegenerative disorders.

Boronate probes for the detection of hydrogen peroxide release from human spermatozoa

Human spermatozoa are compromised by production of reactive oxygen species (ROS), and detection of ROS in spermatozoa is important for the diagnosis of male infertility. The probes 2′,7′-dichlorohydrofluorescein diacetate (DCFH), dihydroethidium (DHE), and MitoSOX red (MSR) are commonly used for detecting ROS by flow cytometry; however, these probes lack sensitivity to hydrogen peroxide (H2O2), which is particularly damaging to mammalian sperm cells. This study reports the synthesis and use of three aryl boronate probes, peroxyfluor-1 (PF1), carboxyperoxyfluor-1, and a novel probe, 2-(2-ethoxyethoxy)ethoxyperoxyfluor-1 (EEPF1), in human spermatozoa. PF1 and EEPF1 were effective at detecting H2O2 and peroxynitrite (ONOO−) produced by spermatozoa when stimulated with menadione or 4-hydroxynonenal. EEPF1 was more effective at detection of ROS in spermatozoa than DCFH, DHE, or MSR; furthermore it distinguished poorly motile sperm as shown by greater ROS production. EEPF1 should therefore have a significant role in the diagnosis of oxidative stress in male infertility, cryopreservation, age, lifestyle, and exposure to environmental toxicants.

The role of hexokinase in cardioprotection - mechanism and potential for translation.

Mitochondrial permeability transition pore (mPTP) opening plays a critical role in cardiac reperfusion injury and its prevention is cardioprotective. Tumour cell mitochondria usually have high levels of hexokinase isoform 2 (HK2) bound to their outer mitochondrial membranes (OMM) and HK2 binding to heart mitochondria has also been implicated in resistance to reperfusion injury. HK2 dissociates from heart mitochondria during ischaemia, and the extent of this correlates with the infarct size on reperfusion. Here we review the mechanisms and regulations of HK2 binding to mitochondria and how this inhibits mPTP opening and consequent reperfusion injury. Major determinants of HK2 dissociation are the elevated glucose‐6‐phosphate concentrations and decreased pH in ischaemia. These are modulated by the myriad of signalling pathways implicated in preconditioning protocols as a result of a decrease in pre‐ischaemic glycogen content. Loss of mitochondrial HK2 during ischaemia is associated with permeabilization of the OMM to cytochrome c, which leads to greater reactive oxygen species production and mPTP opening during reperfusion. Potential interactions between HK2 and OMM proteins associated with mitochondrial fission (e.g. Drp1) and apoptosis (B‐cell lymphoma 2 family members) in these processes are examined. Also considered is the role of HK2 binding in stabilizing contact sites between the OMM and the inner membrane. Breakage of these during ischaemia is proposed to facilitate cytochrome c loss during ischaemia while increasing mPTP opening and compromising cellular bioenergetics during reperfusion. We end by highlighting the many unanswered questions and discussing the potential of modulating mitochondrial HK2 binding as a pharmacological target.Linked ArticlesThis article is part of a themed section on Conditioning the Heart – Pathways to Translation. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue‐8

Glycemic variability and oxidative stress: a link between diabetes and cardiovascular disease?

Diabetes is associated with a two to three-fold increase in risk of cardiovascular disease. However, intensive glucose-lowering therapy aiming at reducing HbA1c to a near-normal level failed to suppress cardiovascular events in recent randomized controlled trials. HbA1c reflects average glucose level rather than glycemic variability. In in vivo and in vitro studies, glycemic variability has been shown to be associated with greater reactive oxygen species production and vascular damage, compared to chronic hyperglycemia. These findings suggest that management of glycemic variability may reduce cardiovascular disease in patients with diabetes; however, clinical studies have shown conflicting results. This review summarizes the current knowledge on glycemic variability and oxidative stress, and discusses the clinical implications.

Age and Heat Exposure-Dependent Changes in Antioxidant Enzymes Activities in Rat's Liver and Brain Mitochondria: Role of α-tocopherol

To investigate the role of mitochondrial antioxidant capacity during increased susceptibility to heat accompanied by the aging, young and aged Wistar rats were exposed on heat for 60 min. After heat exposure, hepatic and brain mitochondria were isolated. Our results revealed changes in antioxidant enzyme activities in liver and brain mitochondria from young and to a greater extent in aged rats. Our measurements of MnSOD, GPx and GR activity indicate greater reactive oxygen species production from the mitochondria of aged heat exposed in comparison to young heat exposed rats. Also in the aged rats, the effect of alpha-tocopherol treatment in the prevention of oxidative stress occurred as a result of heat exposure, is less pronounced. Taken together, our data suggest that mitochondria in aged rats are more vulnerable and less able to prevent oxidative changes that occur in response to acute heat exposure.

The Effect of Pro-Inflammatory Conditioning and/or High Glucose on Telomere Shortening of Aging Fibroblasts

Cardiovascular disease and diabetes have been linked to shorter telomeres, but it is not yet clear which risk factors contribute to shorter telomeres in patients. Our aim was to examine whether pro-inflammatory conditioning, in combination or not with high glucose, result in a higher rate of telomere shortening during in vitro cellular ageing. Human fibroblasts from four donors were cultured for 90 days in: 1) medium lacking ascorbic acid only, 2) 10 mM buthionine sulphoximine (BSO) (pro-oxidant), 3) 25 mM D-glucose, 4) 1 ng/ml IL1B and 5) 25 mM D-glucose+1 ng/ml IL1B. Telomere length was measured with qPCR and intracellular reactive oxygen species (ROS) content and cell death with flow cytometry. Cultures treated with high glucose and BSO displayed a significantly lower growth rate, and cultures treated with IL1B showed a trend towards a higher growth rate, compared to the control [Glucose:0.14 PD/day, p<0.001, BSO: 0.11 PD/day, p = 0.006 and IL1B: 0.19 PD/day, p = 0.093 vs. Control:0.16 PD/day]. Telomere shortening with time was significantly accelerated in cultures treated with IL1B compared to the control [IL1B:−0.8%/day (95%CI:−1.1, −0.5) vs. Control:−0.6%/day (95%CI:−0.8, −0.3), p = 0.012]. The hastening of telomere shortening by IL1B was only in part attenuated after adjustment for the number of cell divisions [IL1B:−4.1%/PD (95%CI:−5.7, −2.4) vs. Control:−2.5%/PD (95%CI:−4.4, −0.7), p = 0.067]. The intracellular ROS content displayed 69% increase (p = 0.033) in BSO compared to the control. In aging fibroblasts, pro-inflammatory conditioning aggravates the shortening of telomeres, an effect which was only in part driven by increased cell turnover. High glucose alone did not result in greater production of ROS or telomere shortening.

Differential responses of antioxidant enzymes to aluminum toxicity in two rice (Oryza sativa L.) cultivars with marked presence and elevated activity of Fe SOD and enhanced activities of Mn SOD and catalase in aluminum tolerant cultivar

Seedlings of two Indica rice (Oryza sativa L.) cvs. HUR-105 and Vandana, differing in Al-tolerance were used to identify the key mechanisms involved in their differential behaviour towards Al toxicity. Cv. HUR-105 appeared to be Al sensitive by showing significant reduction (p ≤ 0.01) in root/shoot length, fresh weight, dry weight and water content in presence of 421 μM Al3+ in growth medium whereas cv. Vandana appeared to be fairly Al3+ tolerant. A conspicuous and significant reduction in dry weight of root and shoot was observed in Al sensitive cv. HUR-105 with 178 μM Al3+ treatment for 3 days. Al was readily taken up by the roots and transported to shoots in both the rice cultivars. Localization of absorbed Al was always greater in roots than in shoots. Our results of the production of reactive oxygen species (ROS) H2O2 and O2.− and activities of major antioxidant enzymes such as total superoxide dismutase (SOD), Cu/Zn SOD, Mn SOD, Fe SOD, catalase (CAT) and guaiacol peroxidase revealed Al induced higher oxidative stress, greater production of ROS and lesser capacity to scavenge ROS in cv. HUR-105 than Vandana. With Al treatment, higher oxidative stress was noted in shoots than in roots. Greatly enhanced activities of SOD (especially Fe and Mn SOD) and CAT in Al treated seedlings of cv. Vandana suggest the role of these enzymes in Al tolerance. Furthermore, a marked presence of Fe SOD in roots and shoots of the seedlings of Al tolerant cv. Vandana and its significant (p ≤ 0.01) increase in activity due to Al-treatment, appears to be the unique feature of this cultivar and indicates a vital role of Fe SOD in Al-tolerance in rice.