Mitofusin 1 and optic atrophy 1 shift metabolism to mitochondrial respiration during aging

Replicative and chronological lifespan are two different modes of cellular aging. Chronological lifespan is defined as the duration during which quiescent normal cells retain their capacity to re‐enter the proliferative cycle. This study investigated whether changes in metabolism occur during aging of quiescent normal human fibroblasts (NHFs) and the mechanisms that regulate these changes. Bioenergetics measurements were taken in quiescent NHFs from younger (newborn, 3‐day, 5‐month, and 1‐year) and older (58‐, 61‐, 63‐, 68‐, and 70‐year) healthy donors as well as NHFs from the same individual at different ages (29, 36, and 46 years). Results show significant changes in cellular metabolism during aging of quiescent NHFs: Old NHFs exhibit a significant decrease in glycolytic flux and lactate levels, and increase in oxygen consumption rate (OCR) and ATP levels compared to young NHFs. Results from the Seahorse XF Cell Mito Stress Test show that old NHFs with a lower Bioenergetic Health Index (BHI) are more prone to oxidative stress compared to young NHFs with a higher BHI. The increase in OCR in old NHFs is associated with a shift in mitochondrial dynamics more toward fusion. Genetic knockdown of mitofusin 1 (MFN1) and optic atrophy 1 (OPA1) in old NHFs decreased OCR and shifted metabolism more toward glycolysis. Downregulation of MFN1 and OPA1 also suppressed the radiation‐induced increase in doubling time of NHFs. In summary, results show that a metabolic shift from glycolysis in young to mitochondrial respiration in old NHFs occurs during chronological lifespan, and MFN1 and OPA1 regulate this process.     

Manganese superoxide dismutase activity regulates transitions between quiescent and proliferative growth

In recent years, the intracellular reactive oxygen species (ROS) levels have gained increasing attention as a critical regulator of cellular proliferation. We investigated the hypothesis that manganese superoxide dismutase (MnSOD) activity regulates proliferative and quiescent growth by modulating cellular ROS levels. Decreasing MnSOD activity favored proliferation in mouse embryonic fibroblasts (MEF), while increasing MnSOD activity facilitated proliferating cells' transitions into quiescence. MnSOD +/- and -/- MEFs demonstrated increased superoxide steady-state levels; these fibroblasts failed to exit from the proliferative cycle, and showed increasing cyclin D1 and cyclin B1 protein levels. MnSOD +/- MEFs exhibited an increase in the percentage of G(2) cells compared to MnSOD +/+ MEFs. Overexpression of MnSOD in MnSOD +/- MEFs suppressed superoxide levels and G(2) accumulation, decreased cyclin B1 protein levels, and facilitated cells' transit into quiescence. While ROS are known to regulate differentiation and cell death pathways, both of which are irreversible processes, our results show MnSOD activity and, therefore, mitochondria-derived ROS levels regulate cellular proliferation and quiescence, which are reversible processes essential to prevent aberrant proliferation and subsequent exhaustion of normal cell proliferative capacity. These results support the hypothesis that MnSOD activity regulates a mitochondrial 'ROS-switch' favoring a superoxide-signaling regulating proliferation and a hydrogen peroxide-signaling supporting quiescence.     

Induction of mitochondrial biogenesis protects against caspase-dependent and caspase-independent apoptosis in L6 myoblasts

Apoptotic signaling plays an important role in skeletal muscle degradation, atrophy, and dysfunction. Mitochondria are central executers of apoptosis by directly participating in caspase-dependent and caspase-independent cell death signaling. Given the important apoptotic role of mitochondria, altering mitochondrial content could influence apoptosis. Therefore, we examined the direct effect of modest, but physiological increases in mitochondrial biogenesis and content on skeletal muscle apoptosis using a cell culture approach. Treatment of L6 myoblasts with SNAP or AICAR (5 h/day for 5 days) significantly increased PGC-1, AIF, cytochrome c, and MnSOD protein content as well as MitoTracker staining. Following induction of mitochondrial biogenesis, L6 myoblasts displayed decreased sensitivity to apoptotic cell death as well as reduced caspase-3 and caspase-9 activation following exposure to staurosporine (STS) and C2-ceramide. L6 myoblasts with higher mitochondrial content also exhibited reduced apoptosis and AIF release following exposure to hydrogen peroxide (H₂O₂). Analysis of several key apoptosis regulatory proteins (ARC, Bax, Bcl-2, XIAP), antioxidant proteins (catalase, MnSOD, CuZnSOD), and reactive oxygen species (ROS) measures (DCF and MitoSOX fluorescence) revealed that these mechanisms were not responsible for the observed cellular protection. However, myoblasts with higher mitochondrial content were less sensitive to Ca^2 +-induced mitochondrial permeability transition pore formation (mPTP) and mitochondrial membrane depolarization. Collectively, these data demonstrate that increased mitochondrial content at physiological levels provides protection against apoptotic cell death by decreasing caspase-dependent and caspase-independent signaling through influencing mitochondrial Ca^2 +-mediated apoptotic events. Therefore, increasing mitochondrial biogenesis/content may represent a potential therapeutic approach in skeletal muscle disorders displaying increased apoptosis.     

Genistein attenuates D-galactose-induced oxidative damage through decreased reactive oxygen species and NF-κB binding activity in neuronal PC12 cells

AimsWe investigated the mechanism of D-galactose (DG)-induced oxidative damage and the neuroprotective action of genistein in PC12 cells.Main methodsPC12 cells were treated with 40 mM DG dissolved in medium containing 85% RPMI1640, 10% HBS and 5% FBS with or without genistein. We measured the protein expression of β-amyloid (Aβ), advanced glycation end products (AGEs), IκB-α and manganese-superoxide dismutase (MnSOD) by western blotting, intracellular reactive oxygen species (ROS) by 2, 7-dichlorofluorescin-diacetate, and the binding activity of nuclear factor kappa B (NF-κB) by electrophortic mobility shift assay.Key findingsDG (40 mM) completely retarded cell growth after incubation for 72 h, and this effect was not due to osmotic changes, as 40 mM mannitol had no effect. Mechanistically, we found that DG increased intracellular ROS starting at 4 h and increased Aβ and AGEs at 24 h. DG treatment for 24 h also increased the binding activity of NF-κB but strongly decreased the expression of IκB-α protein. Furthermore, DG treatment for 48 h increased MnSOD protein expression. All these effects of DG were effectively inhibited by genistein (0.5–10 μM).SignificanceThe present study indicates that the protection of genistein against DG-induced oxidative stress in PC12 cells, and the effect is likely mediated by decreased intracellular ROS and binding activity of NF-κB.     

Melanogenesis inhibits respiration in B16-F10 melanoma cells whereas enhances mitochondrial cell content

Melanoma is a rare and aggressive skin tumor; the survival of patients diagnosed late is fairly low. This high mortality rate is due to the characteristics of the cells that allow them to be resistant to radiotherapy and conventional chemotherapy, besides of being able to evade the immune system. Melanin, the pigment responsible for skin, hair and eye color, seems to be involved in this resistance. The main function of melanin is to protect the cells against ultraviolet (UV) light by absorbing this radiation and reactive oxygen species (ROS) scavenging. But this pigment may have also a role as photosensitizer, because when it is irradiated with UVA light (320-400 nm), the generation of ROS was detected. Besides, the melanogenesis stimulation on B16-F10 cells resulted in cell cycle arrest, induction of a quiescent state, change in the expression of several proteins and alterations on ADP/ATP ratio. The present study aimed to investigate the influence of melanogenesis stimulation in mitochondrial function of B16-F10 melanoma cells. Therefore, we analyzed cells respiration, mitochondrial membrane potential (Δψ_m) and mitochondria mass in B16-F10 melanoma cells stimulated with 0.4 mM L-tyrosine and 10 mM NH₄Cl. Our results showed that the induction of melanin synthesis was able to reduce significantly the oxygen consumption after 48 h of stimulation, without changes of mitochondrial membrane potential when compared to non-stimulated cells. Despite of respiration inhibition, the mitochondria mass was higher in cells with melanogenesis stimulation. We suggest that the stimulation in the melanin synthesis might be promoting the inhibition of electrons transport chain by some intermediate compound from the synthesis of the pigment and this effect could contribute to explain the entry in the quiescent state.     

Persistent damage induces mitochondrial DNA degradation

Considerable progress has been made recently toward understanding the processes of mitochondrial DNA (mtDNA) damage and repair. However, a paucity of information still exists regarding the physiological effects of persistent mtDNA damage. This is due, in part, to experimental difficulties associated with targeting mtDNA for damage, while sparing nuclear DNA. Here, we characterize two systems designed for targeted mtDNA damage based on the inducible (Tet-ON) mitochondrial expression of the bacterial enzyme, exonuclease III, and the human enzyme, uracil-N-glyosylase containing the Y147A mutation. In both systems, damage was accompanied by degradation of mtDNA, which was detectable by 6 h after induction of mutant uracil-N-glycosylase and by 12 h after induction of exoIII. Unexpectedly, increases in the steady-state levels of single-strand lesions, which led to degradation, were small in absolute terms indicating that both abasic sites and single-strand gaps may be poorly tolerated in mtDNA. mtDNA degradation was accompanied by the loss of expression of mtDNA-encoded COX2. After withdrawal of the inducer, recovery from mtDNA depletion occurred faster in the system expressing exonuclease III, but in both systems reduced mtDNA levels persisted longer than 144 h after doxycycline withdrawal. mtDNA degradation was followed by reduction and loss of respiration, decreased membrane potential, reduced cell viability, reduced intrinsic reactive oxygen species production, slowed proliferation, and changes in mitochondrial morphology (fragmentation of the mitochondrial network, rounding and “foaming” of the mitochondria). The mutagenic effects of abasic sites in mtDNA were low, which indicates that damaged mtDNA molecules may be degraded if not rapidly repaired. This study establishes, for the first time, that mtDNA degradation can be a direct and immediate consequence of persistent mtDNA damage and that increased ROS production is not an invariant consequence of mtDNA damage.     

Redox-Optimized ROS Balance and the relationship between mitochondrial respiration and ROS

The Redox-Optimized ROS Balance [R-ORB] hypothesis postulates that the redox environment [RE] is the main intermediary between mitochondrial respiration and reactive oxygen species [ROS]. According to R-ORB, ROS emission levels will attain a minimum vs. RE when respiratory rate (VO₂) reaches a maximum following ADP stimulation, a tenet that we test herein in isolated heart mitochondria under forward electron transport [FET]. ROS emission increased two-fold as a function of changes in the RE (~ 400 to ~ 900 mV·mM) in state 4 respiration elicited by increasing glutamate/malate (G/M). In G/M energized mitochondria, ROS emission decreases two-fold for RE ~ 500 to ~ 300 mV·mM in state 3 respiration at increasing ADP. Stressed mitochondria released higher ROS, that was only weakly dependent on RE under state 3. As a function of VO₂, the ROS dependence on RE was strong between ~ 550 and ~ 350 mV·mM, when VO₂ is maximal, primarily due to changes in glutathione redox potential. A similar dependence was observed with stressed mitochondria, but over a significantly more oxidized RE and ~ 3-fold higher ROS emission overall, as compared with non-stressed controls. We conclude that under non-stressful conditions mitochondrial ROS efflux decreases when the RE becomes less reduced within a range in which VO₂ is maximal. These results agree with the R-ORB postulate that mitochondria minimize ROS emission as they maximize VO₂ and ATP synthesis. This relationship is altered quantitatively, but not qualitatively, by oxidative stress although stressed mitochondria exhibit diminished energetic performance and increased ROS release.     

Human mitochondrial haplogroup H: The highest VO2max consumer – Is it a paradox?

Mitochondrial background has been demonstrated to influence maximal oxygen uptake (VO_2max, in mL kg^?1 min^?1), but this genetic influence can be compensated for by regular exercise. A positive correlation among electron transport chain (ETC) coupling, ATP and reactive oxygen species (ROS) production has been established, and mitochondrial variants have been reported to show differences in their ETC performance. In this study, we examined in detail the VO_2max differences found among mitochondrial haplogroups. We recruited 81 healthy male Spanish Caucasian individuals and determined their mitochondrial haplogroup. Their VO_2max was determined using incremental cycling exercise (ICE). VO_2max was lower in J than in non-J haplogroup individuals (P = 0.04). The H haplogroup was responsible for this difference (VO_2max; J vs. H; P = 0.008) and this group also had significantly higher mitochondrial oxidative damage (mtOD) than the J haplogroup (P = 0.04). In agreement with these results, VO_2max and mtOD were positively correlated (P = 0.01). Given that ROS production is the major contributor to mtOD and consumes four times more oxygen per electron than the ETC, our results strongly suggest that ROS production is responsible for the higher VO_2max found in the H variant. These findings not only contribute to a better understanding of the mechanisms underneath VO_2max, but also help to explain some reported associations between mitochondrial haplogroups and mtOD with longevity, sperm motility, premature aging and susceptibility to different pathologies.     

Effect of thermal stress on HSP70 expression in dermal fibroblast of zebu (Tharparkar) and crossbred (Karan-Fries) cattle

The present studies were conducted to investigate the difference response of dermal fibroblasts to heat stress in Tharparkar and Karan-Fries cattle. Skin is the most important environmental interface providing a protective envelope to animals. In skin, dermal fibroblasts are the most regular cell constituent of dermis that is crucial for temperature homeostasis. The study aimed to examine the reactive oxygen species (ROS) formation, cytotoxicity (%) and heat shock protein 70 (HSP70) genes expression in dermal fibroblast of Tharparkar and Karan-Fries cattle and to assess whether resistance of dermal fibroblast to heat stress is breed specific. Dermal fibroblasts from ear pinna of Tharparkar and Karan-Fries cattle were exposed at 25 °C, 37 °C, 40 °C and 44 °C for 3 h to measure the ROS, cytotoxicity (%) and HSP 70 (HSPA1A, HSPA2 and HSPA8) genes’ expression. The results showed that ROS formation at low temperature (25 °C) decreased in both breeds as compared to control (37 °C) and the differences were significant (P<0.0001). Heat stress at 40 °C did not increase ROS formation significantly in Tharparkar but increased significantly (P<0.001) in Karan-Fries cattle. The overall cytotoxicity (%) was also found to be significantly different (P<0.001) between Tharparkar and Karan-Fries cattle, and on exposure to different temperatures (P<0.001). The cytotoxicity (%) in dermal fibroblast cells of Karan-fries cows was more than Tharparkar. The expression studies indicated that all HSP70 genes (HSPA8, HSPA1A and HSPA2) were up-regulated at different temperatures in both breeds. In Tharparkar, the relative mRNA expression of HSPA8 gene was higher but HSPA1A and HSPA2 genes were low as compared to Karan-Fries cattle. At 40 and 44 °C, the relative expressions of inducible HSP 70 genes (HSPA1A and HSPA2) were higher in Karan-Fries than Tharparkar. In summary, dermal fibroblast resistance to heat shock differed between breeds. Dermal fibroblasts of Tharparkar were observed to be more heat tolerant than crossbred Karan-Fries cattle. The study concludes that zebu cattle (Tharparkar) dermal fibroblasts are more adapted to tropical climatic condition than crossbreed cattle (Karan-Fries). Differences exist in dermal fibroblasts of heat adapted and non-adapted cattle.     

Cardiac mitochondrial biogenesis in endotoxemia is not accompanied by mitochondrial function recovery

Mitochondrial biogenesis emerges as a compensatory mechanism involved in the recovery process in endotoxemia and sepsis. The aim of this work was to analyze the time course of the cardiac mitochondrial biogenesis process occurring during endotoxemia, with emphasis on the quantitative analysis of mitochondrial function. Female Sprague-Dawley rats (45 days old) were ip injected with LPS (10 mg/kg). Measurements were performed at 0–24 h after LPS administration. PGC-1α and mtTFA expression for biogenesis and p62 and LC3 expression for autophagy were analyzed by Western blot; mitochondrial DNA levels by qPCR, and mitochondrial morphology by transmission electron microscopy. Mitochondrial function was evaluated as oxygen consumption and respiratory chain complex activity. PGC-1α and mtTFA expression significantly increased in every time point analyzed, and mitochondrial mass was increased by 20% (P<0.05) at 24 h. p62 expression was significantly decreased in a time-dependent manner. LC3-II expression was significantly increased at all time points analyzed. Ultrastructurally, mitochondria displayed several abnormalities (internal vesicles, cristae disruption, and swelling) at 6 and 18 h. Structures compatible with fusion/fission processes were observed at 24 h. A significant decrease in state 3 respiration was observed in every time point analyzed (LPS 6 h: 20%, P<0.05). Mitochondrial complex I activity was found decreased by 30% in LPS-treated animals at 6 and 24 h. Complex II and complex IV showed decreased activity only at 24 h. The present results show that partial restoration of cardiac mitochondrial architecture is not accompanied by improvement of mitochondrial function in acute endotoxemia. The key implication of our study is that cardiac failure due to bioenergetic dysfunction will be overcome by therapeutic interventions aimed to restore cardiac mitochondrial function.     

‘Mild mitochondrial uncoupling’ induced protection against neuronal excitotoxicity requires AMPK activity

The preconditioning response conferred by a mild uncoupling of the mitochondrial membrane potential (Δψ_m) has been attributed to altered reactive oxygen species (ROS) production and mitochondrial Ca^2 + uptake within the cells. Here we have explored if altered cellular energetics in response to a mild mitochondrial uncoupling stimulus may also contribute to the protection. The addition of 100 nM FCCP for 30 min to cerebellar granule neurons (CGNs) induced a transient depolarization of the Δψ_m, that was sufficient to significantly reduce CGN vulnerability to the excitotoxic stimulus, glutamate. On investigation, the mild mitochondrial ‘uncoupling’ stimulus resulted in a significant increase in the plasma membrane levels of the glucose transporter isoform 3, with a hyperpolarisation of Δψ_m and increased cellular ATP levels also evident following the washout of FCCP. Furthermore, the phosphorylation state of AMP-activated protein kinase (AMPK) (Thr 172) was increased within 5 min of the uncoupling stimulus and elevated up to 1 h after washout. Significantly, the physiological changes and protection evident after the mild uncoupling stimulus were lost in CGNs when AMPK activity was inhibited. This study identifies an additional mechanism through which protection is mediated upon mild mitochondrial uncoupling: it implicates increased AMPK signalling and an adaptive shift in energy metabolism as mediators of the preconditioning response associated with FCCP-induced mild mitochondrial uncoupling.     

How do reactive oxygen species and calcium trigger mitochondrial membrane permeabilisation?

BackgroundMitochondrial membrane permeabilisation (MMP) is classically considered as a point of no return in several forms of cell death and is involved in numerous diseases such as cancer, neurodegenerative disorders or ischemia/reperfusion injuries. Many studies established that reactive oxygen species (ROS) and Ca^2 + were the prominent inducers of MMP. However, the mechanisms connecting ROS and Ca^2 + to the players of MMP are still a matter of debate.Scope of reviewThe aim of this review is to summarise the various studies related to the mechanisms of ROS- and Ca^2 +-induced MMP. Several lines of evidence suggest that ROS and Ca^2 + cooperate to induce MMP but the molecular details of the ROS–Ca^2 +-MMP network remain controversial. We then discuss recent data depicting this topic.Major conclusionsCytotoxic stimuli may be transduced within the cell by ROS and Ca^2 + increases. In most models, Ca^2 + and ROS can cooperate to induce MMP. Moreover, several data suggest that MMP increases mitochondrial Ca^2 + and ROS which therefore amplify the cytotoxic signal. Intriguingly, many reports have identified players of MMP as direct ROS targets. On the contrary, direct targets of Ca^2 + remain elusive. At the same time, the mechanisms by which mitochondrial Ca^2 + overload induces ROS generation are well documented. Upon these observations, we hypothesise that Ca^2 + cannot directly induce MMP and requires ROS production as a mandatory step.General significanceGiven the importance of Ca^2 +- and ROS-induced MMP in diseases, we expect that a better understanding of this process will lead to the development of novel therapies.     

Cellulose nanocrystal cationic derivative induces NLRP3 inflammasome-dependent IL-1β secretion associated with mitochondrial ROS production

Crystalline cellulose nanocrystals (CNCs) have emerged as novel materials for a wide variety of important applications such as nanofillers, nanocomposites, surface coatings, regenerative medicine and potential drug delivery. CNCs have a needle-like structure with sizes in the range of 100–200 nm long and 5–20 nm wide and a mean aspect ratio 10–100. Despite the great potential applicability of CNCs, very little is known about their potential immunogenicity. Needle-like materials have been known to evoke an immune response in particular to activate the (NOD-like receptor, pyrin domain-containing 3)-inflammasome/IL-1β (Interleukin 1β) pathway. In this study we evaluated the capacity of unmodified CNC and its cationic derivatives CNC-AEM (aminoethylmethacrylate)1, CNC-AEM2, CNC-AEMA(aminoethylmethacrylamide)1 and CNC-AEMA2 to stimulate NLRP3-inflammasome/IL-1β pathway and enhance the production of mitochondrial reactive oxygen species (ROS). Mouse macrophage cell line (J774A.1) was stimulated for 24 h with 50 µg/mL with unmodified CNC and its cationic derivatives. Alternatively, J774A1 or PBMCs (peripheral blood mononuclear cells) were stimulated with CNC-AEMA2 in presence or absence of LPS (lipopolysaccharide). IL-1β secretion was analyzed by ELISA, mitochondrial function by JC-1 staining and ATP content. Intracellular and mitochondrial reactive oxygen species (ROS) were assessed by DCF-DA (2′,7′-dichlorodihydrofluorescein diacetate) and MitoSOX, respectively. Mitochondrial ROS and extracellular ATP were significantly increased in cells treated with CNC-AEMA2, which correlates with the strongest effects on IL-1β secretion in non-primed cells. CNC-AEMA2 also induced IL-1βsecretion in LPS-primed and non-primed PBMCs. Our data suggest that the increases in mitochondrial ROS and ATP release induced by CNC-AEMA2 may be associated with its capability to evoke immune response. We demonstrate the first evidence that newly synthesized cationic cellulose nanocrystal derivative, CNC-AEMA2, has immunogenic properties, which may lead to the development of a potential non-toxic and safe nanomaterial to be used as a novel adjuvant for vaccines.     

The mitochondria-targeted anti-oxidant MitoQ reduces aspects of mitochondrial fission in the 6-OHDA cell model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder for which available treatments provide symptom relief but do not stop disease progression. Mitochondria, and in particular mitochondrial dynamics, have been postulated as plausible pharmacological targets. Mitochondria-targeted antioxidants have been developed to prevent mitochondrial oxidative damage, and to alter the involvement of reactive oxygen species (ROS) in signaling pathways. In this study, we have dissected the effect of MitoQ, which is produced by covalent attachment of ubiquinone to a triphenylphosphonium lipophilic cation by a ten carbon alkyl chain. MitoQ was tested in an in vitro PD model which involves addition of 6-hydroxydopamine (6-OHDA) to SH-SY5Y cell cultures. At sublethal concentrations of 50 μM, 6-OHDA did not induce increases in protein carbonyl, mitochondrial lipid peroxidation or mitochondrial DNA damage. However, after 3 h of treatment, 6-OHDA disrupts the mitochondrial morphology and activates the machinery of mitochondrial fission, but not fusion. Addition of 6-OHDA did not increase the levels of fission 1, mitofusins 1 and 2 or optic atrophy 1 proteins, but does lead to the translocation of dynamin related protein 1 from the cytosol to the mitochondria. Pre-treatment with MitoQ (50 nM, 30 min) results in the inhibition of the mitochondrial translocation of Drp1. Furthermore, MitoQ also inhibited the translocation of the pro-apoptotic protein Bax to the mitochondria. These findings provide mechanistic evidence for a role for redox events contributing to mitochondrial fission and suggest the potential of mitochondria-targeted therapeutics in diseases that involve mitochondrial fragmentation due to oxidative stress.     

Endurance training and chronic intermittent hypoxia modulate in vitro salicylate-induced hepatic mitochondrial dysfunction

Mitochondrial function is modulated by multiple approaches including physical activity, which can afford cross-tolerance against a variety of insults. We therefore aimed to analyze the effects of endurance-training (ET) and chronic-intermittent hypobaric-hypoxia (IHH) on liver mitochondrial bioenergetics and whether these effects translate into benefits against in vitro salicylate mitochondrial toxicity.Twenty-eight young-adult male rats were divided into normoxic-sedentary (NS), normoxic-exercised (NE), hypoxic-sedentary (HS) and hypoxic-exercised (HE). ET consisted of 1 h/days of treadmill running and IHH of simulated atmospheric pressure of 49.3 kPa 5 h/days during 5 weeks. Liver mitochondrial oxygen consumption, transmembrane-electric potential (ΔΨ) and permeability transition pore induction (MPTP) were evaluated in the presence and absence of salicylate. Aconitase, MnSOD, caspase-3 and 8 activities, SH, MDA, SIRT3, Cyp D, HSP70, and OXPHOS subunit contents were assessed.ET and IHH decreased basal mitochondrial state-3 and state-4 respiration, although no alterations were observed in ΔΨ endpoints evaluated in control mitochondria. In the presence of salicylate, ET and IHH decreased state-4 and lag-phase of ADP-phosphorylation. Moreover, ADP-lag phase in hypoxic was further lower than in normoxic groups. Neither ET nor IHH altered the susceptibility to calcium-induced MPTP. IHH lowered MnSOD and increased aconitase activities. ET and IHH decreased caspase 8 activity whereas no effect was observed on caspase 3. The levels of SIRT3 increased with ET and IHH and Cyp D decreased with IHH.Data suggest that ET and IHH do not alter general basal liver mitochondrial function, but may attenuate some adverse effects of salicylate.     

Changes in rat spermatozoa function after cooling,cryopreservation and centrifugation processes

Rat sperm cryopreservation is an effective method of archiving valuable strains for biomedical research and handling of rat spermatozoa is very important for successful cryopreservation. The aim of this study was to evaluate changes in rat sperm function during cryopreservation and centrifugation. Epididymal rat spermatozoa were subjected to cooling and freezing–thawing processes and then motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were compared before and after minimum centrifugation force (200×g). Cryopreservation decreased sperm motility, PMI, and MMP (P < 0.05). Basal (without ROS inducer, tert-butyl hydroperoxide [TBHP] treatment) and stimulated ROS (with TBHP treatment) were increased in viable cooled spermatozoa compared to viable fresh spermatozoa (P < 0.01), with equal susceptibility to TBHP among fresh, cooled, and frozen–thawed spermatozoa. Centrifugation decreased motility and PMI of frozen–thawed spermatozoa (P < 0.05). Centrifugation decreased basal ROS of all spermatozoa (P < 0.01), while it led to higher susceptibility to TBHP in viable cooled spermatozoa, showing higher increased fold in ROS and decreased rate in viability by TBHP in viable cooled spermatozoa (P < 0.05). Cooling process was the major step of ROS generation, with loss in sperm motility, PMI, and MMP. Centrifugation affected function of cryopreserved spermatozoa. These data suggest that centrifugation makes rat spermatozoa susceptible to external ROS source, in particular during cooling process. Thus, protection from ROS damage and minimizing centrifugation should be considered during cryopreservation and post-thaw use of cryopreserved epididymal rat spermatozoa.     

Mitochondrial calcium uniporter blocker effectively prevents brain mitochondrial dysfunction caused by iron overload

AimsAlthough iron overload induces oxidative stress and brain mitochondrial dysfunction, and is associated with neurodegenerative diseases, brain mitochondrial iron uptake has not been investigated. We determined the role of mitochondrial calcium uniporter (MCU) in brain mitochondria as a major route for iron entry. We hypothesized that iron overload causes brain mitochondrial dysfunction, and that the MCU blocker prevents iron entry into mitochondria, thus attenuating mitochondrial dysfunction.Main methodsIsolated brain mitochondria from male Wistar rats were used. Iron (Fe^2 + and Fe^3 +) at 0–286 μM were applied onto mitochondria at various incubation times (5–30 min), and the mitochondrial function was determined. Effects of MCU blocker (Ru-360) and iron chelator were studied.Key findingsBoth Fe^2 + and Fe^3 + entered brain mitochondria and caused mitochondrial swelling in a dose- and time-dependent manner, and caused mitochondrial depolarization and increased ROS production. However, Fe^2 + caused more severe mitochondrial dysfunction than Fe^3 +. Although all drugs attenuated mitochondrial dysfunction caused by iron overload, only an MCU blocker could completely prevent ROS production and mitochondrial depolarization.SignificanceOur findings indicated that iron overload caused brain mitochondrial dysfunction, and that an MCU blocker effectively prevented this impairment, suggesting that MCU could be the major portal for brain mitochondrial iron uptake.     

Characterization of cytotoxicity induced by arsenic trioxide (a potent anti-APL drug) in rat cardiac myocytes

Arsenic, a known environmental toxicant, is ubiquitously present in the environment. Arsenic trioxide (ATO), an anti-acute promyelocytic leukemia (APL) drug, is associated with cardiac toxicity. It is reported to induce cardiac arrhythmia via altering various ion channels involved in the repolarization phase of cardiac action potential. The exact molecular mechanism of cardiovascular adverse effect due to ATO exposure has not been fully elucidated except for alteration on ion channels. To evaluate the cytotoxic effect of ATO on cardiac myocytes, primary culture of myocytes was treated with different doses (30, 60 and 90 μM) of ATO for various periods (24, 48 and 72 h). Cardiac toxicity was assessed by monitoring cell viability, mitochondrial and deoxyribonucleic acid (DNA) integrity, reactive oxygen species (ROS) generation, calcium overload and apoptosis. ATO exposure caused alteration in mitochondrial integrity, generation of ROS, calcium overload and apoptosis in cardiac cells in dose- and duration-dependent manner. There was no DNA fragmentation. Hence our results show that ATO causes apoptosis in cardiomyocytes by generation of ROS and the induction of calcium overload.