mROS Formation Research Articles

Role of Mitochondrial Reactive Oxygen Species-Mediated Chaperone-Mediated Autophagy and Lipophagy in Baicalin and N-Acetylcysteine Mitigation of Cadmium-Induced Lipid Accumulation in Liver.

Cadmium (Cd) is a major health concern globally and can accumulate and cause damage in the liver for which there is no approved treatment. Baicalin and N-acetylcysteine (NAC) have been found to have protective effects against a variety of liver injuries, but it is not clear whether their combined use is effective in preventing and treating Cd-induced lipid accumulation. The study found that Cd increased the production of mitochondrial reactive oxygen species (mROS) and elevated the level of chaperone-mediated autophagy (CMA). Interestingly, mROS-mediated CMA exacerbates the Cd-induced inhibition of lipophagy. Baicalin and NAC counteracted inhibition of lipophagy by attenuating Cd-induced CMA, suggesting an interplay between CMA elevation, mitochondrial destruction, and mROS formation. Maintaining the stability of mitochondrial structure and function is essential for alleviating Cd-induced lipid accumulation in the liver. Choline is an essential component of the mitochondrial membrane and is responsible for maintaining its structure and function. Mitochondrial transcriptional factor A (TFAM) is involved in mitochondrial DNA transcriptional activation and replication. Our study revealed that the combination of baicalin and NAC can regulate choline metabolism through TFAM and thereby maintain mitochondrial structure and functionality. In summary, the combination of baicalin and NAC plays a more beneficial role in alleviating Cd-induced lipid accumulation than the drug alone, and the combination of baicalin and NAC can stabilize mitochondrial structure and function and inhibit mROS-mediated CMA through TFAM-choline, thereby promoting lipophagy to alleviate Cd-induced lipid accumulation.

Temporal correlation of morphological and biochemical changes with the recruitment of different mechanisms of reactive oxygen species formation during human SW872 cell adipogenic differentiation.

Human SW872 preadipocyte conversion to mature adipocytes is associated with time‐dependent changes in differentiation markers' expression and with morphological changes accompanied by the accumulation of lipid droplets (LDs) as well as by increased mitochondriogenesis and mitochondrial membrane potential. Under identical conditions, the formation of reactive oxygen species (ROS) revealed with a general probe was significant at days 3 and 10 of differentiation and bearly detectable at day 6. NADPH oxidase (NOX)‐2 activity determined with an immunocytochemical approach followed a very similar pattern. There was no evidence of mitochondrial ROS (mROS), as detected with a selective fluorescence probe, at days 3 and 6, possibly due to the triggering of the Nrf‐2 antioxidant response. mROS were instead clearly detected at day 10, concomitantly with the accumulation of very large LDs, oxidation of both cardiolipin and thioredoxin 2, and decreased mitochondrial glutathione. In conclusion, the morphological and biochemical changes of differentiating SW872 cells are accompanied by the discontinuous formation of ROS derived from NOX‐2, increasingly implicated in adipogenesis and adipose tissue dysfunction. In addition, mROS formation was significant only in the late phase of differentiation and was associated with mitochondrial dysfunction.

MCU-Dependent mROS Generation Regulates Cell Metabolism and Cell Death Modulated by the AMPK/PGC-1α/SIRT3 Signaling Pathway

The mitochondrial calcium uniporter is an intensively investigated calcium channel, and its molecular components, structural features, and encoded genes have long been explored. Further studies have shown that the mitochondrial calcium unidirectional transporter (MCU) is a macromolecular complex related to intracellular and extracellular calcium regulation. Based on the current understanding, the MCU is crucial for maintaining cytosolic Ca2+ (cCa2+) homeostasis by modulating mitochondrial Ca2+ (mCa2+) uptake. The elevation of MCU-induced calcium levels is confirmed to be the main cause of mitochondrial reactive oxygen species (mROS) generation, which leads to disordered cellular metabolic patterns and cell death. In particular, in an I/R injury model, cancer cells, and adipocytes, MCU expression is maintained at high levels. As is well accepted, the AMPK/PGC-1α/SIRT3 pathway is believed to have an affinity for mROS formation and energy consumption. Therefore, we identified a link between MCU-related mROS formation and the AMPK/PGC-1α/SIRT3 signaling pathway in controlling cell metabolism and cell death, which may provide a new possibility of targeting the MCU to reverse relevant diseases.

An ex vivo Approach to Assess Mitochondrial ROS by Flow Cytometry in AAV-tagged Astrocytes in Adult Mice.

Mitochondrial reactive oxygen species (mROS) are naturally produced signalling molecules extremely relevant for understanding both health- and disease-associated biological processes. The study of mROS in the brain is currently underway to decipher their physiopathological roles and contributions in neurological diseases. Recent advances in this field have highlighted the importance of studying mROS signalling and redox biology at the cellular level. Neurons are especially sensitive to the harmful effects of excess mROS while astrocytic mROS have been shown to play a relevant physiological role in cerebral homeostasis and behaviour. However, given the complexity of the brain, investigating mROS formation in a specific cell-type in adult animals is methodologically challenging. Here we propose an approach to specifically assess mROS abundance in astrocytes that combines i) a targeting strategy based on the use of adeno-associated virus (AAV) vectors expressing the green fluorescent protein (GFP) under an astrocyte (glial fibrillary acidic protein or GFAP) promoter, along with, ii) a robust and widely extended protocol for the measurement of mROS by flow cytometry using commercial probes. The significance of this work is that it allows the selective study of astrocytic mROS abundance by means of easily accessible technology.

The interplay between mitochondrial reactive oxygen species formation and the coenzyme Q reduction level

Our aim was to elucidate the relationship between the rate of mitochondrial reactive oxygen species (mROS) formation and the reduction level of the mitochondrial coenzyme Q (mQ) pool under various levels of engagement of the mQ-reducing pathway (succinate dehydrogenase, complex II) and mQH2-oxidizing pathways (the cytochrome pathway and alternative oxidase pathway, (AOX)) in mitochondria isolated from the amoeba Acanthamoeba castellanii. The mQ pool was shifted to a more reduced state by inhibition of mQH2-oxidizing pathways (complex III and complex IV of the cytochrome pathway, and AOX) and the oxidative phosphorylation system. The mQ reduction level was lowered by decreasing the electron supply from succinate dehydrogenase and by stimulating the activity of the cytochrome or AOX pathways. The results indicate a direct dependence of mROS formation on the reduction level of the mQ pool for both mQH2-oxidizing pathways. A higher mQ reduction level leads to a higher mROS formation. For the cytochrome pathway, mROS generation depends nonlinearly upon the mQ reduction level, with a stronger dependency observed at values higher than the mQ reduction level of the phosphorylating state (~ 35%). AOX becomes more engaged at higher mQ pool reduction levels (above 40%), when mROS production via the cytochrome pathway increases. We propose that the mQ pool reduction level (endogenous mQ redox state) could be a useful endogenous reporter that allows indirect assessment of overall mROS production in mitochondria.

MtDNA T8993G Mutation-Induced F1F0-ATP Synthase Defect Augments Mitochondrial Dysfunction Associated with hypoxia/reoxygenation: The Protective Role of Melatonin

BackgroundF1F0-ATP synthase (F1F0-ATPase) plays important roles in regulating mitochondrial function during hypoxia, but the effect of F1F0-ATPase defect on hypoxia/reoxygenation (H/RO) is unknown. The aim of this study was to investigate how mtDNA T8993G mutation (NARP)-induced inhibition of F1F0-ATPase modulates the H/RO–induced mitochondrial dysfunction. In addition, the potential for melatonin, a potent antioxidant with multiple mitochondrial protective properties, to protect NARP cells exposed to H/RO was assessed.Methods And FindingsNARP cybrids harboring 98% of mtDNA T8993G genes were established as an in vitro model for cells with F1F0-ATPase defect; their parental osteosarcoma 143B cells were studied for comparison. Treating the cells with H/RO using a hypoxic chamber resembles ischemia/reperfusion in vivo. NARP significantly enhanced apoptotic death upon H/RO detected by MTT assay and the trypan blue exclusion test of cell viability. Based on fluorescence probe-coupled laser scanning imaging microscopy, NARP significantly enhanced mitochondrial reactive oxygen species (mROS) formation and mitochondrial Ca2+ (mCa2+) accumulation in response to H/RO, which augmented the depletion of cardiolipin, resulting in the retardation of mitochondrial movement. With stronger H/RO stress (either with longer reoxygenation duration, longer hypoxia duration, or administrating secondary oxidative stress following H/RO), NARP augmented H/RO-induced mROS formation to significantly depolarize mitochondrial membrane potential (ΔΨm), and enhance mCa2+ accumulation and nitric oxide formation. Also, NARP augmented H/RO-induced mROS oxidized and depleted cardiolipin, thereby promoting permanent mitochondrial permeability transition, retarded mitochondrial movement, and enhanced apoptosis. Melatonin markedly reduced NARP-augmented H/RO-induced mROS formation and therefore significantly reduced mROS-mediated depolarization of ΔΨm and accumulation of mCa2+, stabilized cardiolipin, and then improved mitochondrial movement and cell survival. ConclusionNARP-induced inhibition of F1F0-ATPase enhances mROS formation upon H/RO, which augments the depletion of cardiolipin and retardation of mitochondrial movement. Melatonin may have the potential to rescue patients with ischemia/reperfusion insults, even those associated with NARP symptoms.

Dual Phases of Respiration Chain Defect-Augmented mROS-Mediated mCa2+Stress during Oxidative Insult in Normal andρ0RBA1 Astrocytes

Mitochondrial respiratory chain (RC) deficits, resulting in augmented mitochondrial ROS (mROS) generation, underlie pathogenesis of astrocytes. However, mtDNA-depleted cells (ρ 0) lacking RC have been reported to be either sensitive or resistant to apoptosis. In this study, we sought to determine the effects of RC-enhanced mitochondrial stress following oxidative insult. Using noninvasive fluorescence probe-coupled laser scanning imaging microscopy, the ability to resist oxidative stress and levels of mROS formation and mitochondrial calcium (mCa2+) were compared between two different astrocyte cell lines, control and ρ 0 astrocytes, over time upon oxidative stress. Our results showed that the cytoplasmic membrane becomes permeated with YO-PRO-1 dye at 150 and 130 minutes in RBA-1 and ρ 0 astrocytes, respectively. In contrast to RBA-1, 30 minutes after 20 mM H2O2 exposure, ρ 0 astrocytes formed marked plasma membrane blebs, lost the ability to retain Mito-R, and showed condensation of nuclei. Importantly, H2O2-induced ROS and accompanied mCa2+ elevation in control showed higher levels than ρ 0 at early time point but vice versa at late time point. Our findings underscore dual phase of RC-defective cells harboring less mitochondrial stress due to low RC activity during short-term oxidative stress but augmented mROS-mediated mCa2+ stress during severe oxidative insult.

Long‐term Aβ exposure augments mCa2+‐independent mROS‐mediated depletion of cardiolipin for the shift of a lethal transient mitochondrial permeability transition to its permanent mode in NARP cybrids: a protective targeting of melatonin

Mitochondrial dysfunction is a hallmark of amyloid β-peptide (Aβ)-induced neurodegeneration of Alzheimer's disease (AD). This study investigated whether mtDNA T8993G mutation-induced complex V inhibition, clinically associated with neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP), is a potential risk factor for AD and the pathological link for long-term exposure of Aβ-induced mitochondrial toxicity and apoptosis in NARP cybrids. Using noninvasive fluorescence probe-coupled laser scanning imaging microscopy and NARP cybrids harboring 98% mutant genes along with its parental 143B osteosarcoma cells, we demonstrated that Aβ-augmented mitochondrial Ca(2+) (mCa(2+))-independent mitochondrial reactive oxygen species (mROS) formation for a cardiolipin (CL, a major mitochondrial protective phospholipid)-dependent lethal modulation of the mitochondrial permeability transition (MPT). Aβ augmented not only the amount but also the propagation rate of mROS-induced mROS formation to significantly depolarize mitochondrial membrane potential (∆Ψ(m)) and reduce mCa(2+) stress. Aβ-augmented mROS oxidized and depleted CL, thereby enhances mitochondrial fission and movement retardation, which promoted the NARP-augmented lethal transient-MPT (t-MPT) to switch to its irreversible mode of permanent-MPT (p-MPT). Interestingly, melatonin, a multiple mitochondrial protector, markedly reduced Aβ-augmented mROS formation and therefore significantly reduced mROS-mediated depolarization of ∆Ψ(m), fission of mitochondria and retardation of mitochondrial movement to stabilize CL and hence the MPT. In the presence of melatonin, Aβ-promoted p-MPT was reversed to a protective t-MPT, which preserved ∆Ψ(m) and lowered elevated mCa(2+) to sublethal levels for an enhanced mCa(2+)-dependent O(2) consumption. Thus, melatonin may potentially rescue AD patients associated with NARP symptoms.

Increased Mitochondrial Sensitivity To Proton Leak Is Responsible For Decreased Reactive Oxygen Species Production In Ischemic Preconditioning

Introduction: Mitochondrial reactive oxygen species (mROS) cause significant damage to myocardium after an episode of ischemia-reperfusion (IR). Ischemic preconditioning (IPC) decreases mROS formation and protects myocardium from IR. The mechanism of decreased mROS in IPC is unknown. Proton leak (H+ leak) dissipates mitochondria membrane potential (mΔΨ) and may regulate mROS production by changing the redox status of mitochondria. We hypothesize that increased H+ leak is responsible for decreased mROS production during IPC. Methods: Rat hearts were assigned to CONTROL (30 min of equilibration (EQ), 30 min ischemia (I), and 30 min of reperfusion (RP)) or IPC (10 min EQ, two 5 min episodes of IR, 30 min I, and 30 min RP). Rate pressure product (RPP) was measured throughout. Mitochondria were isolated at end equilibration (end EQ), end ischemia (end I), and end reperfusion (end RP). Respiration was measured by polarography, and mΔΨ with a TPP electrode. H+ leak is the respiratory rate required to maintain mΔΨ of -130mV in the presence of oligomycin-A, and was titrated with FCCP (0-160nM). Complex III mROS production was measured by fluorometry using Amplex-Red at end EQ and by electron paramagnetic resonance at end I and end RP. Results: Induction of IPC increased mROS production at end EQ (59±2 vs. 43±2 nmoles H2O2/mg protein/min, p<0.05, Fig.1) but did not change H+ leak (37±9 vs. 30±10 nmoles O/mg protein/min) when compared to CONTROL. IPC attenuated the increase in H+ leak at end I seen in CONTROL (44±11 vs. 140±11 nmoles O/mg protein/min, p<0.001). At end RP, IPC decreased mROS production (2115±146 vs. 3277±333 arbitrary units/mg protein, p<0.05). There was no difference in H+ leak between IPC and CONTROL at end RP (34±9 vs. 45±9 nmoles O/mg protein/min). IPC mitochondria responded to depolarization with greater reduction in mROS production than CONTROL (Fig.1). Further depolarization led to a trend of increased mROS in both CONTROL and IPC. The inflection point occurred at a higher mΔΨ in IPC mitochondria (-160 vs. -138 mV; Fig. 1). Conclusions: Increased rate of H+ leak is not the mechanism of decreased mROS after IR. While IPC did not lead to increased H+ leak, the induction of IPC increases the sensitivity of mitochondria to changes in mΔΨ leading to greater reduction in mROS per unit of change in mΔΨ. This increased sensitivity explains the greater reduction in mROS at end RP despite a lower H+ leak. Reducing mROS formation after ischemia-reperfusion is important to improving outcomes in patients with acute coronary syndrome.

Cryopreservation-induced alterations in boar spermatozoa mitochondrial function are related to changes in the expression and location of midpiece mitofusin-2 and actin network

The authors analyzed changes in mitochondrial activity of boar semen during a standard cryopreservation protocol. For this purpose, mitochondrial activity was evaluated simultaneously with the rhythm of mitochondrial formation of reactive oxygen species (mROS) through a double MitoTracker Red/proxylfluorescamine stain. Moreover, we analyzed changes in the expression and location of two key regulatory elements of mitochondrial function, namely mitofusin-2 (Mfn2) and actin, during the freezing-thawing protocol. Our results indicate that mitochondrial activity and mROS formation decreased during cyropreservation, with an initial decrease during the cooling phase of the protocol. This decrease was accompanied by an increase in the amount of solubilized Mfn2, which was concomitant with a progressive extension of Mfn2 location from the apical zone of the midpiece to the whole midpiece. Simultaneously, cryopreservation induced a decrease in solubilized actin, which was concurrent with significant changes in the midpiece actin location. The observed changes in the expression and location of both Mfn2 and actin were already present after the cooling phase of the cryopreservation protocol. Our results suggest that freezing-thawing impaired mitochondrial function. This impairment was concomitant with a decrease in the mitochondrial capacity to synthesize mROS. This impairment is attributed to changes in mitochondrial volume as a result of alterations in the expression and location of both Mfn-2 and the actin network. Finally, the alterations of mitochondrial function induced by the cryopreservation protocol were already apparent at the cooling phase. This observation indicates that the cooling phase is a crucial stage in which mitochondrial alterations occur during cryopreservation.

MtDNA T8993G-Augmented Mitochondrial Stresses Upon mCa2+ Overload and its Protection by Melatonin in a NARP Cybrid

Mitochondrial DNA (mtDNA) T8993G mutation inhibits specifically mitochondrial F1F0-ATPase (complex V) for severe ATP deficiency and is clinically associated with neurological muscle weakness, ataxia, and retinitis pigmentosa so called NARP mutation. At present, detail T8993G-associated mitochondrial mechanisms as well as its therapeutic strategies are limited. Using time-lapse laser scanning dual fluorescence imaging microscopy, this study investigated T8993G-altered apoptotic mitochondrial pathology particular upon mCa2+ stress and protection by melatonin, previously reported to protect mCa2+ stress-mediated apoptosis (Hsu et al., 2009 JPR in press). In comparison to its parental osteosarcoma 143B and mtDNA less (ρ0) cells, T8993G induced significant hyperpolarization of mitochondrial membrane potential (ΔΨm) and potentiated greatly ionomycin-induced mCa2+ stress. T8993G-augmented mCa2+ stress subsequently elicited rigorously generation of mitochondrial oxygen species (mROS), depletion of cardiolipin (CL) and activation of the mitochondrial permeability transition (MPT). In contract, ρ0 cells, with much depolarized ΔΨm, suffered less mCa2+ stress, mROS formation, CL depletion and the MPT opening. Interestingly, melatonin reduced significantly peak amplitude of the ionomycin-induced mCa2+ transient and antagonized efficiently mCa2+-augmented mROS generation for a reduced depletion of CL and activation of the MPT. In addition, melatonin prevented “oxidation free mCa2+”-mediated MPT suggesting its direct targeting on the MPT. Melatonin-enhanced tail amplitude of mCa2+ transient possibly due to the reduced MPT-dependent depolarization of ΔΨm, however, did not enhance mCa2+ stress-mediated pathology in NARP cybrids possibly as melatonin-elevated mCa2+ improved mitochondrial respiratory. Thus, the administration of melatonin may provide potential improvement for the treatment of mtDNA T8993G-associated NARP syndromes and diseases.

Visualization of Melatonin's Multiple Mitochondrial Levels of Protection Against Mitochondrial Ca2+-Mediated Permeability Transition and Beyond in Rat Brain Astrocytes

Melatonin protects cells against oxidative stress-induced apoptosis due primarily to its ability to effectively scavenge pathological condition-augmented generation of mitochondrial reactive oxygen species (mROS). Once produced, mROS in addition to indiscriminately damage mitochondrial components they crucially activate directly the mitochondrial permeability transition (MPT), one of the critical mechanisms for initiating post mitochondrial apoptotic signaling. Whether or not melatonin targets directly the MPT, however, remains inconclusive, particularly during oxidative stress. Thus, we investigated this possibility of an “oxidation free Ca2+ stress” in the presence of vitamin E after ionomycin exposure as a sole Ca2+-mediated MPT in order to exclude melatonin's primary antioxidative effects as well as Ca2+-mediated oxidative stress. With the application of laser scanning fluorescence imaging microscopy, we visualized for the first time multiple mitochondrial protections provided by melatonin during Ca2+ stress in cultured rat brain astrocytes RBA-1. Melatonin, due to its primary antioxidative actions, completely prevented mCa2+-induced mROS formation for a reduced mROS-activated MPT during ionomycin exposure. In the presence of vitamin E, melatonin, significantly reduced cyclosporin A (CsA) sensitive mitochondrial depolarization and MPT during ionomycin exposure suggesting its direct targeting of the MPT. Moreover, when the MPT was inhibited by CsA, melatonin reduced further MPT-independent mitochondrial depolarization and apoptosis suggesting its targeting beyond the MPT. As astrocytes play active role in regulating neuronal pathophysiology, these multiple mitochondrial protections provided by melatonin against mCa2+- and/or mROS-mediated apoptosis may thus be crucial for the future therapeutic prevention and treatment of astrocyte-mediated neurodegeneration in the CNS.

Visualization of melatonin’s multiple mitochondrial levels of protection against mitochondrial Ca2+‐mediated permeability transition and beyond in rat brain astrocytes

Melatonin protects cells against various types of oxidative stress-induced apoptosis due primarily to its ability to effectively scavenge pathological and disease condition-augmented generation of mitochondrial reactive oxygen species (mROS). Once produced, mROS indiscriminately damage mitochondrial components and more importantly they crucially activate directly the mitochondrial permeability transition (MPT), one of the critical mechanisms for initiating post mitochondrial apoptotic signaling. Whether or not melatonin targets directly the MPT, however, remains inconclusive, particularly during oxidative stress. This study, thus, investigated this possibility of an 'oxidation free Ca(2+) stress' in the presence of vitamin E after ionomycin exposure as a sole Ca(2+)-mediated MPT in order to exclude melatonin's primary antioxidative effects as well as Ca(2+)-mediated oxidative stress. The studies were carried out using cultured rat brain astrocytes RBA-1. With the application of laser scanning multiple fluorescence imaging microscopy, we visualized for the first time multiple mitochondrial protective effects provided by melatonin during Ca(2+) stress. First, melatonin, due to its primary antioxidative actions, completely prevented mCa(2+)-induced mROS formation during ionomycin exposure. Secondly, when melatonin(')s antioxidative effects were prevented due to the addition of vitamin E, melatonin significantly prevented mCa(2+)-mediated MPT and apoptosis suggesting its direct targeting of the MPT. Surprisingly, in the presence of cyclosporin A, a MPT inhibitor, melatonin reduced further mCa(2+)-mediated apoptosis during ionomycin exposure also suggesting its targeting beyond the MPT. As astrocytes are actively involve in regulating synaptic transmission and neurovascular coupling in the CNS, these multiple mitochondrial layers of protection provided by melatonin against mCa(2+)-and/or mROS-mediated apoptosis in astrocytes may be crucial for future therapeutic prevention and treatment of astrocyte-mediated neurodegenerative diseases in the CNS.

Visualization Of Mitochondria-targeted Photodynamic Effects Of Hpph-in Coupled With Visible Laser 637 Nm In Osteosarcoma 143b Cells

Photosensitizer HPPH cooperated with metal complex containing In (III) (HPPH-In) produces much efficient singlet oxygen production and photodynamic effects (PDE) as compared to HPPH alone. With the application of mitochondrial fluorescent probes and laser scanning imaging microscopy, mitochondrial level of PDE induced by HPPH-In coupled with visible laser 637 nm were investigated in detail. PDE of HPPH-In significantly enhances depletion of a mitochondria specific fluorescent probe MitoTracker Green at very earlier time points suggesting its primary targeting on the mitochondrial membrane. Mitochondria soon swelled and followed by plasma membrane blebing and later apoptotic condensation of nuclei and cell death. These mitochondria-associated apoptotic events induced by PDE of HPPH-In can be partially inhibited by a mitochondria antioxidant, MitoQ, and by the removal of extracellular Ca2+ suggesting a mROS- and Ca2+-dependent mechanism is involved. When mitochondrial reactive oxygen species (ROS) formation and mitochondrial membrane potential depolarization (ΔΨ) were imaged simultaneously, PDE of HPPH-In significantly enhanced mROS formation and ΔΨ depolarization with small delay. PDE of HPPH-In-induced increase in mROS soon propagated to adjacent non-irradiated mitochondrial population as well as that in adjacent cells and caused depolarization ofΔΨ of these non-irradiated mitochondria. In addition to PDE of HPPH-In-enhanced mROS formation, we observed PDE of HPPH-In-induced sudden depolarization of ΔΨeffectively reduced mROS formation suggesting a possible protective preconditioning may exist. Finally, PDE of HPPH-In significantly altered mitochondrial dynamics by decreasing mitochondrial movement and enhancing fission of mitochondria. These observations suggest that multiple mitochondria-targeted devastating mechanisms provided by the PDE of HPPH-In coupled with 637 nm laser may be particularly useful in the eradication of malignant tumors for cancer therapy.

Melatonin protects against common deletion of mitochondrial DNA‐augmented mitochondrial oxidative stress and apoptosis

Defected mitochondrial respiratory chain (RC), in addition to causing a severe ATP deficiency, often augments reactive oxygen species (ROS) generation in mitochondria (mROS) which enhances pathological conditions and diseases. Previously, we demonstrated a potent endogenously RC defect-augmented mROS associated dose-dependently with a commonly seen large-scale deletion of 4977 base pairs of mitochondrial DNA (mtDNA), i.e. the common deletion (CD). As current treatments for CD-associated diseases are rather supplementary and ineffective, we investigated whether melatonin, a potential mitochondrial protector, provides beneficial protection for CD-augmented mitochondrial oxidative stress and apoptosis particularly upon the induction of a secondary oxidative stress. Detailed mechanistic investigations were performed by using laser scanning dual fluorescence imaging microscopy to provide precise spatial and temporal resolution of mitochondrial events at single cell level. We demonstrate, for the first time, that melatonin significantly prevents CD-augmented mROS formation under basal conditions as well as at early time-points upon secondary oxidative stress induced by H2O2 exposure. Thus, melatonin prevents mROS-mediated depolarization of mitochondrial membrane potential (DeltaPsim) and subsequent opening of the mitochondrial permeability transition pore (MPTP) and cytochrome c release. Moreover, melatonin prevents depletion of cardiolipin which appears to be crucial for postponing later MPTP opening, disruption of the mitochondrial membrane and apoptosis. Finally, the protection provided by melatonin is superior to those caused by the suppression of mitochondrial Ca2+ regulators including the mitochondrial Na+-Ca2) exchanger, the MPTP, and the mitochondrial Ca2+ uniporter and by antioxidants including vitamin E and mitochondria-targeted coenzyme Q, MitoQ. As RC defect-augmented endogenous mitochondrial oxidative stress is centrally involved in a variety of pathological conditions and diseases, melatonin thus may serve as a therapeutic drug to benefit many clinical conditions that involve malfunction of the mitochondria.

Photosensitizer Targeting: Mitochondrion-Targeted Photosensitizer Enhances Mitochondrial Reactive Oxygen Species and Mitochondrial Calcium-Mediated Apoptosis

Using laser scanning imaging microscopy, our previous studies have demonstrated visible laser irradiation induced significant generation of mitochondrial reactive oxygen species (mROS) and apoptosis. In addition, photodynamic therapy (PDT) with a mitochondrion-targeted photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), significantly enhanced visible laser irradiation induced mitochondrial dysfunction and apoptosis. To validate the critical role of the mitochondrion as well as how mitochondrial mechanisms were involved in the PDT of BPD-MA, subcellular PDT with BPD-MA were performed using region irradiation and recorded by multi-photon imaging microscopy coupled with mitochondrial specific fluorescent probes in single live C6 glioma cells. Specifically, mROS and mitochondrial free calcium (mCa2+) were simultaneously measured from cells received subcellular PDT with BPD-MA in different compartments including the mitochondrion, the cytosol and the nucleus. Results revealed that significant formation of mROS accompanied by elevation of mCa2+ was found only in the mitochondrial PDT with BPD-MA. Subsequently, mitochondrial PDT with BPD-MA resulted in severe mitochondrial swelling, plasma membrane blebing and apoptotic death. We conclude that via augmented mROS formation and mCa2+ elevation PDT with a mitochondrion-targeted photosensitizer extensively enhances mitochondria-mediated apoptosis and, thus, may provide high eradication efficacy for clinical cancer treatment.

Mitochondrion‐Targeted Photosensitizer Enhances the Photodynamic Effect‐Induced Mitochondrial Dysfunction and Apoptosis

Recently, the mitochondrion has been considered as a novel pharmacological target for anticancer therapy due to its crucial role involved in arbitrating cell apoptosis. We have previously demonstrated that 488-nm laser irradiation induced a specific mitochondrial reactive oxygen species (mROS) formation and apoptotic death. In this study, we used a second generation of photosensitizers, the benzoporphyrin-derivative monoacid ring A (BPD-MA). We investigated specifically mechanisms at the mitochondrial level for BPD-MA coupled with 690-nm laser irradiation, the photodynamic effect (PDE) of BPD-MA, using conventional and laser scanning imaging microscopy in intact C6 glioma cells. We demonstrated BPD-MA localized mainly in the mitochondrial area. The phototoxicity induced by 1-10 J 690-nm laser irradiation was minor as compared to that induced by 488-nm laser irradiation. Unlike other mitochondrion-targeted photosensitizers, the dark toxicity induced by BPD-MA (0.05-5 mg/mL, effective doses used for the PDE) was relatively low. Nevertheless, the PDE of BPD-MA using 0.5 mg/mL coupled with 5J 690-nm irradiation induced profound and rapid (< 1 min) mitochondrial swelling, mROS formation, and severe plasma membrane blebbing as compared to that induced by 488-nm laser irradiation (< 10 min). Later, the PDE of BPD-MA resulted in positive propidium iodide cell-death stain and positive TUNEL apoptotic nuclear stain and DNA laddering. Finally, the PDT of BPD-MA also instantaneously promoted the mitochondrion to diminish its covalent binding with a mitochondrial marker, MitoTracker Green. We conclude that the PDT of BPD-MA targeted primarily and compellingly the mitochondrion to induce effective mitochondria-mediated apoptosis and thus may serve as a powerful photosensitizer for clinical cancer therapy.

Mitochondrial Reactive Oxygen Species Generation and Calcium Increase Induced by Visible Light in Astrocytes

Abstract: Mitochondria contain photosensitive chromophores that can be activated or inhibited by light in the visible range. Rather than utilizing light energy, however, mitochondrial electron transport oxidation‐reduction reaction and energy coupling could be stimulated or damaged by visible light. Our previous work demonstrated that reactive oxygen species (ROS) were generated in cultured astrocytes after visible laser irradiation. With confocal fluorescence microscopy, we found that ROS were generated mostly from mitochondria. This mitochondrial ROS (mROS) formation plays a critical role in photoirradiation‐induced phototoxicity and apoptosis. In this study, we measured changes of mitochondrial calcium level ([Ca2+]m) in cultured astrocytes (RBA‐1 cell line) irradiated with blue light and examined the association between mROS formation and [Ca2+]m level changes. Changes of intracellular ROS and [Ca2+]m were visualized using fluorescent probes 2′,7′‐dichlorodihydrofluorescein (DCF), and rhod‐2 After exposure to visible light irradiation, RBA‐1 astrocytes showed a rapid increase in ROS accumulation particularly in the mitochondrial area. Increase in [Ca2+]m was also induced by photoirradiation. The levels of increase in DCF fluorescence intensity varied among different astrocytes. Some of the cells generated much higher levels of ROS than others. For those cells that had high ROS levels, mitochondrial Ca2+ levels were also high. In cells that had mild ROS levels, mitochondrial Ca2+ levels were only slightly increased. The rate of increase in DCF fluorescence seemed to be close to the rate of rhod‐2 fluorescence increase. There is a positive and close correlation between mitochondrial ROS levels and mitochondrial Ca2+ levels in astrocytes irradiated by visible light.

Mitochondrial Swelling and Generation of Reactive Oxygen Species Induced by Photoirradiation Are Heterogeneously Distributed

Abstract: Abundant evidence has been gathered to show that overproduction of reactive oxygen species (ROS) can lead to the opening of the mitochondrial permeability transition pore (MPTP) and result in apoptosis in mammalian cells. The information regarding spatial and temporal regulation of intracellular ROS formation related to the MPTP opening, however, is relatively limited. In this study, we used a fluorescent probe, dihydro‐2′,7′‐dichloroforescin (DCF), to detect intracellular ROS levels in different compartments of the cell in a time‐resolved manner. The roles of mitochondrial ROS (mROS) in the MPTP opening and mitochondrial membrane potential drop were investigated by using H2DCFDA coloaded with a mitochondrial marker dye MitoTracker Red, and by a mitochondrial membrane potential dye tetramethyl rhodamine ethyl ester. We applied multiphoton laser scanning microscopy to avoid autooxidation and bleaching of DCF so that long‐term visualization of intracellular ROS formation could be performed. Moreover, we noted that the resting mROS levels of different mitochondria were not homogeneous. After cells had been exposed to photoirradiation, the intracellular ROS gradually increased but the heterogeneity of mROS was maintained. Later, swelling was observed in mitochondria that contained higher levels of ROS, indicating the opening of the MPTP. In cells in which all the mitochondria swelled, they were translocated to the perinuclear area, which became the site of ROS production. At this stage, mROS reached the highest level concomitantly with a complete loss of mitochondrial membrane potential, indicating full opening of the MPTP. At the end, photoirradiation resulted in apoptotic cell death. In summary, we demonstrated by multiphoton laser scanning microscopy that photoirradiation induces heterogeneous intracellular ROS formation and mitochondrial permeability transition pore opening in single intact cells. These observations imply the existence of a microdomain in the regulation of mROS formation and subsequent opening of the MPTP.

Mitochondrial reactive oxygen species generation and calcium increase induced by visible light in astrocytes.

Mitochondria contain photosensitive chromophores that can be activated or inhibited by light in the visible range. Rather than utilizing light energy, however, mitochondrial electron transport oxidation-reduction reaction and energy coupling could be stimulated or damaged by visible light. Our previous work demonstrated that reactive oxygen species (ROS) were generated in cultured astrocytes after visible laser irradiation. With confocal fluorescence microscopy, we found that ROS were generated mostly from mitochondria. This mitochondrial ROS (mROS) formation plays a critical role in photoirradiation-induced phototoxicity and apoptosis. In this study, we measured changes of mitochondrial calcium level ([Ca(2+)](m)) in cultured astrocytes (RBA-1 cell line) irradiated with blue light and examined the association between mROS formation and [Ca(2+)](m) level changes. Changes of intracellular ROS and [Ca(2+)](m) were visualized using fluorescent probes 2',7'-dichlorodihydrofluorescein (DCF), and rhod-2. After exposure to visible light irradiation, RBA-1 astrocytes showed a rapid increase in ROS accumulation particularly in the mitochondrial area. Increase in [Ca(2+)](m) was also induced by photoirradiation. The levels of increase in DCF fluorescence intensity varied among different astrocytes. Some of the cells generated much higher levels of ROS than others. For those cells that had high ROS levels, mitochondrial Ca(2+) levels were also high. In cells that had mild ROS levels, mitochondrial Ca(2+) levels were only slightly increased. The rate of increase in DCF fluorescence seemed to be close to the rate of rhod-2 fluorescence increase. There is a positive and close correlation between mitochondrial ROS levels and mitochondrial Ca(2+) levels in astrocytes irradiated by visible light.