Photobiomodulation on isolated mitochondria at 810 nm: first results on the efficiency of the energy conversion process.

In this paper the photobiomodulation on isolated mitochondria of bovine liver is studied as a thermodynamic process of conversion of energy. This analysis is conducted by considering a particular set-up for the photobiomodulation experiments of interest. It allows, in particular, the computation of the electromagnetic field and the related energetic quantities in the stimulated organelles. The measurements of the excess of biochemical power density produced by the illuminated mitochondria are performed at regular time intervals after the experiments. The calculations and the measurements finally allow us to obtain the first results on the efficiency of the process of conversion of electromagnetic energy into excess of biochemical energy released by the isolated organelles.

Berberine, a natural alkaloid sensitizes human hepatocarcinoma to ionizing radiation by blocking autophagy and cell cycle arrest resulting in senescence.

OBJECTIVE: To study the radiosensitizing potential of Berberine and the underlying mechanism in human hepatocarcinoma (HepG2) cells. METHODS: HepG2 cells were challenged with X-rays in combination with Berberine treatment and several in vitro assays were performed. Alteration in cell viability was determined by MTT assay. Changes in intracellular ROS levels, mitochondrial membrane potential/mass, intracellular acidic vesicular organelles as well as cell cycle arrest and apoptotic cell death were analysed by flow cytometry. Induction of autophagy was assessed by staining the cells with Monodansylcadaverine/Lysotracker red dyes and immunoblotting for LC3I/II and p62 proteins. Phase-contrast/fluorescence microscopy was employed to study mitotic catastrophe and senescence. Cellular senescence was confirmed by immunoblotting for p21 levels and ELISA for Interleukin-6. KEY FINDINGS: X-rays + Berberine had a synergistic effect in reducing cell proliferation accompanied by a robust G2/M arrest. Berberine-mediated radiosensitization was associated with elevated levels of LC3II and p62 suggesting blocked autophagy that was followed by mitotic catastrophe and senescence. Treatment of cells with X-rays + Berberine resulted in increased oxidative stress, hyperpolarized mitochondria with increased mitochondrial mass and reduced ATP levels. CONCLUSIONS: The study expands the understanding of the pharmacological properties of Berberine and its applicability as a radiosensitizer towards treating liver cancer.

A mitohormetic response to pro-oxidant exposure in the house mouse.

Mitochondria are hypothesized to display a biphasic response to reactive oxygen species (ROS) exposure. In this study, we evaluated the time course changes in mitochondrial performance and oxidative stress in house mice following X-irradiation. Forty-eight mice were equally divided among six groups, including a nonirradiated control and five experimental groups that varied in time between X-ray exposure and euthanasia (1 h and 1, 4, 7, and 10 days after X-irradiation). We measured parameters associated with mitochondrial respiratory function and ROS emission from isolated liver and skeletal muscle mitochondria and levels of oxidative damage and antioxidants in liver, skeletal muscle, and heart tissues. Mitochondrial function dropped initially after X-irradiation but recovered quickly and was elevated 10 days after the exposure. Hydrogen peroxide production, lipid peroxidation, and protein carbonylation showed inverse U-shaped curves, with levels returning to control or lower than control, 10 days after X-irradiation. Enzymatic antioxidants and markers for mitochondrial biogenesis exhibited a tissue-specific response after irradiation. These data provide the first chronological description of the mitohormetic response after a mild dose of irradiation and highlight the protective response that cells display to ROS exposure. This study also provides valuable information and application for future mitochondrial and oxidative stress studies in numerous physiological settings.

Effect of irradiation with different laser wavelengths on oxidative stress of non-hepatectomized rats.

PURPOSE: To assess the effect of two laser wavelengths, either separate or combined, on intact rat livers. METHOD: Nineteen male Wistar rats (200-300 g) were submitted to laser irradiation at 5 different sites on the liver surface.Wavelengths 660 and 780 nm were used, with a dose of irradiation of 60 J/cm2/site.The animals were divided into the groups:control (C) and animals irradiated with 660 nm laser (L1), with 780 nm laser (L2) or withboth wavelengths (L3).Mitochondrial function, mitochondrial swelling, and hepatocellular malondialdehyde (MDA) levels were determined.Data were analyzed by the Mann-Whitney test, with the level of significance set at 5%. RESULTS: There was a reduction of ADP-activated respiration (state 3) in group L1 compared to group C (p=0.0016), whereas the values of group L2 were similar to control.Group L3 also showed a reduction of state 3 (p=0.0159).There was a reduction of RCR in group L1 compared to control (p=0.0001) and to group L2 (p=0.0040).Mitochondrial swelling only differed between group L3 and control (p=0.0286).There was a increase in MDA levels in group L3 compared to control (p=0.0476) and to group L2 (p=0.0286) and in group L1 compared to group L2 (p=0.0132). CONCLUSION: Although laser irradiation reduced mitochondrial function,it did not interfere with the hepatocellular energy status.

Effect of irradiation with different laser wavelengths on oxidative stress of non-hepatectomized rats

PURPOSE: To assess the effect of two laser wavelengths, either separate or combined, on intact rat livers. METHOD: Nineteen male Wistar rats (200-300 g) were submitted to laser irradiation at 5 different sites on the liver surface.Wavelengths 660 and 780 nm were used, with a dose of irradiation of 60 J/cm2/site.The animals were divided into the groups:control (C) and animals irradiated with 660 nm laser (L1), with 780 nm laser (L2) or withboth wavelengths (L3).Mitochondrial function, mitochondrial swelling, and hepatocellular malondialdehyde (MDA) levels were determined.Data were analyzed by the Mann-Whitney test, with the level of significance set at 5%. RESULTS: There was a reduction of ADP-activated respiration (state 3) in group L1 compared to group C (p=0.0016), whereas the values of group L2 were similar to control.Group L3 also showed a reduction of state 3 (p=0.0159).There was a reduction of RCR in group L1 compared to control (p=0.0001) and to group L2 (p=0.0040).Mitochondrial swelling only differed between group L3 and control (p=0.0286).There was a increase in MDA levels in group L3 compared to control (p=0.0476) and to group L2 (p=0.0286) and in group L1 compared to group L2 (p=0.0132). CONCLUSION: Although laser irradiation reduced mitochondrial function,it did not interfere with the hepatocellular energy status.

Protection of radiation induced DNA and membrane damages by total triterpenes isolated from Ganoderma lucidum (Fr.) P. Karst.

The total triterpenes isolated from the fruiting bodies of Ganoderma lucidum was examined for its potential to prevent γ-radiation induced membrane damage in rat liver mitochondria and microsomes. The effects of total triterpenes on γ-radiation-induced DNA strand breaks in pBR 322 plasmid DNA in vitro and human peripheral blood lymphocytes ex vivo were evaluated. The protective effect of total triterpenes against γ-radiation-induced micronuclei formations in mice bone marrow cells in vivo were also evaluated. The results indicated the significant effectiveness of Ganoderma triterpenes in protecting the DNA and membrane damages consequent to the hazardous effects of radiation. The findings suggest the potential use of Ganoderma triterpenes in radio therapy.

Are the mitochondrial respiratory complexes blocked by NO the targets for the laser and LED therapy?

Effects of laser (442 and 532 nm) and light-emitting diode (LED) (650 nm) radiation on mitochondrial respiration and mitochondrial electron transport rate (complexes II-III and IV) in the presence of nitric oxide (NO) were investigated. It was found that nitric oxide (300 nM-10 µM) suppresses mitochondrial respiration. Laser irradiation of mitochondria (442 nm, 3 J cm(-2)) partly restored mitochondrial respiration (approximately by 70 %). Irradiation with green laser (532 nm) or red LED (650 nm) in the same dose had no reliable effect. Evaluation of mitochondrial electron transport rate in complexes II-III and IV and effects of nitric oxide demonstrated almost similar sensitivity of complex II-III and IV to NO, with approximately 50 % inhibition at NO concentration of 3 µM. Subsequent laser or LED irradiation (3 J cm(-2)) showed partial recovery of electron transport only in complex IV and only under irradiation with blue light (442 nm). Our results support the hypothesis of the crucial role of cytochrome c oxidase (complex IV) in photoreactivation of mitochondrial respiration suppressed by NO.

Reversible effects of photodamage directed toward mitochondria.

When the initial effect of photodynamic therapy (PDT) involves mitochondrial photodamage, an early effect is loss of the mitochondrial membrane potential (ΔΨm ). Using murine hepatoma 1c1c7 cells and a photosensitizing agent known to target mitochondria, we examined loss of ΔΨm , initiation of apoptosis and loss of viability as a function of time and light dose. There was a correlation between loss of viability and the rapid disappearance of ΔΨm, as detected by the potential-sensitive probe Mitotracker Orange (MTO). Loss of ΔΨm was, however, reversible even with a substantial loss of viability. Unless there was a supralethal level of photodamage, 1c1c7 cells recovered their mitochondrial membrane potential, even if the cell population was on the pathway to apoptosis and cell death. These results indicate that when mitochondria are the initial PDT target, a qualitative estimate of photokilling can be provided by assessing the initial loss of ΔΨm.

Protective effect of Cichorium glandulosum seeds from ultraviolet B-induced damage in rat liver mitochondria.

Cichorium glandulosum Boiss. et Huet, a common herb for treating hepatitis, is indigenous to Europe, Western Asia, and the Xinjiang Uygur Autonomous Region of China. This study aims at evaluating the protective activity of different extracts from C. glandulosum seeds against experimental oxidation- and ultraviolet B (UVB)-induced damage in rat liver mitochondria. The antioxidant property of different extracts from C. glandulosum seeds was investigated by employing various established in vitro systems, such as α,α-diphenyl-ß-picrylhydrazyl, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid), and reducing power assay. The protective effects of different C. glandulosum seed extracts against UVB-induced phototoxicity in a mitochondria model were also evaluated by measuring thiobarbituric acid reactive substances, glutathione, lipid hydroperoxide, conjugated diene, and 4-hydroxynonenal. The main compounds in C. glandulosum seeds were identified by HPLC-PDA-ESI-MS/MS. The results showed that C. glandulosum seed extracts have strong antioxidant activity, in which the ethyl acetate extract (EE) and n-butanol extract (BE) showed better activity than other extracts. In a UVB-induced mitochondria model, both EE and BE have better antioxidant activity and protective effects against phototoxicity than the petroleum ether extract, chloroform extract, and water extract. The differences in antioxidant activity and photoprotective capacity among these five extracts are associated with their phenolic compound content. Therefore, research on this function of C. glandulosum seeds may broaden their applications in the food and medical industry.

Carbon ion beams induce hepatoma cell death by NADPH oxidase-mediated mitochondrial damage.

Mitochondria are a major source of reactive oxygen species (ROS) and are also the target of cellular ROS. ROS damage to mitochondria leads to dysfunction that further enhances the production of mitochondrial ROS. This feed-forward vicious cycle between mitochondria and ROS induces cell death. Within a few minutes of radiation exposure, NADPH oxidase is activated to elevate the ROS level. Activated NADPH oxidase might induce the feed-forward cycle of mitochondria and this is a possible mechanism for cancer cell death induced by heavy ion irradiation. We found that after 4 Gy of (12) C(6+) ion radiation of HepG2 cells, the NADPH oxidase membrane subunit gp91(phox) was not involved in enzyme activation through increased expression; however, the subunit p47(phox) was involved in activation by being translocated to the membrane. (12) C(6+) ion radiation clearly decreased the ΔΨm of HepG2 cells, increasing mitochondrial DNA damage and inducing cell death. Pretreatment with apocynin (APO, an NADPH oxidase inhibitor) effectively prevented the ΔΨm decrease, mitochondrial DNA damage, and cell death induced by radiation. However, these protective effects were not observed with APO treatment after irradiation exposure. These data demonstrated that NADPH oxidase activation was an initiator in mitochondrial damage. Once mitochondria entered the feed-forward cycle, cell fate was no longer controlled by NADPH oxidase. Only antioxidants that targeted mitochondria such as MitoQ could break the cycle and release cells from death.

Maternal bias in mouse radiosensitivity: the role of the mitochondrial PTP.

This study investigated, at the molecular level, mitochondrial responses to radiation. In three mouse strains, we found the following: (1) mitochondrial response to calcium stress was associated with a strain's susceptibility to γ-radiation; (2) γ-radiation increased this calcium stress response in a dose-responsive manner; (3) the mitochondrial DNA (mtDNA) copy number in the liver of the radiosensitive mouse strain was significantly lower, as compared to that of the radioresistant strain; (4) adenine nucleotide translocase (ANT) mRNA copy numbers were significantly lower in the radiosensitive strain; (5) the F1 offspring (BC/C57M) of radiosensitive females mated with radioresistant males exhibited a significant difference in calcium stress response from that of the radiation-resistant strain, but the reverse cross did not exhibit this difference; and (6) only those mitochondria extracted from the livers of irradiated BC/C57M mice exhibited a heightened calcium stress response. We propose that a genetic change in ANT and a postirradiation change involving either mtDNA-encoded protein replacement or altered mtDNA association fit these data.

[The antioxidant enzyme activity in mouse liver mitochondria after nanosecond pulsed periodic X-ray exposure].

The effect of repetitive pulsed X-ray (4 ns pulse duration, 300 kV accelerating voltage; 2.5 kA electron beam current) on the antioxidant enzyme activity in mouse liver mitochondria has been investigated. The mitochondrial suspension was exposed to single 4000 pulse X-ray radiation with repetition rates ranging between 10 and 22 pps (pulsed dose was 0.3-1.8 x 10(-6) Gy/pulse, the total absorbed dose following a single exposure was 7.2 x 10(-3) Gy). It was shown that a short-time exposure to X-ray radiation changes the antioxidant enzyme activity in mouse liver mitochondria. The greatest effect was observed in the changes of the activity of the metal-containing enzymes: superoxide dismutase and glutathione peroxidase. The effect depends on the pulse repetition frequency and radiation dose.

In vitro and in vivo evaluation of inhibition activity of lotus (Nelumbo nucifera Gaertn.) leaves against ultraviolet B-induced phototoxicity.

Lotus (Nelumbo nucifera Gaertn.), an aquatic vegetable, is extensively cultivated in eastern Asia, particularly in China. Our previous study showed that lotus leaf extracts (LLEs) have strong antioxidant effects in vitro and in vivo. The main antioxidants in lotus leaf have been identified via liquid chromatography-mass spectrometry. Ultraviolet B (UVB) protective effects have been associated with plant extracts rich in antioxidants. The current study focuses on the mitochondria model to evaluate the potent inhibition activity of LLE against UVB-induced phototoxicity. The level of thiobarbituric acid reactive substances, glutathione, lipid hydroperoxide, conjugated diene, and 4-hydroxynonenal were measured. The in vivo activity of LLE was also investigated in mice model. The results showed that all concentrations of LLE (10, 100, and 1000µg/ml) possessed strong protective effect against UVB-induced phototoxicity in the mitochondria model. The in vivo test showed that LLE have significant protective effects on the level of superoxide dismutase, catalase, and glutathione peroxidase, as well as the contents of hydroxyproline and malondialdehyde in the skin samples. This study would provide a foundation for broadening the applications of lotus leaf in both the medical and food industries.

Electromagnetic pulse reduces free radical generation in rat liver mitochondria in vitro.

Non-ionizing radiation electromagnetic pulse (EMP) is generally recorded to induce the generation of free radicals in vivo. Though mitochondria are the primary site to produce free radicals, a rare report is designed to directly investigate the EMP effects on free radical generation at mitochondrial level. Thus the present work was designed to study how EMP induces free radical generation in rat liver mitochondria in vitro using electron paramagnetic resonance technique. Surprisingly, our data suggest that EMP prevents free radical generation by activating antioxidant enzyme activity and reducing oxygen consumption and therefore free radical generation. Electron spin resonance measurements clearly demonstrate that disordering of mitochondrial lipid fluidity and membrane proteins mobility are the underlying contributors to this decreased oxygen consumption. Therefore, our results suggest that EMP might hold the potentiality to be developed as a non-invasive means to benefit certain diseases.

[The state of the cytochrome part of respiratory chain in tumor carrier rats' liver in the conditions of preliminary low-level irradiation].

The effect of low-level irradiation on the structural and functional organization of the cytochrome part of the respiratory chain in tumor carrier rats' liver is studied. The preliminary low-level irradiation leading to the mitochondrial cytochrome a, b and c content reduction at the latent stage of Guerin's carcinoma is shown. At the same time, the maximal reduction of the content of all liver cytochromes is observed at the terminal stages of oncogenesis. The content of cytochome c undergoes the most significant changes in the liver mitochondrial fracture. The possible mechanism of mitochondrial haem-containing cytochromes content reduction may be associated with the disorder of their formation caused by the heam synthesis inhibition found in our study. Simultaneously, the cytochrome oxydase (key enzyme of the cytochrome part) activity inhibition is observed to be caused by preliminary low-level irradiation at the latent growth stage of Guerin's carcinoma. The determined differences between irradiated and non-irradiated tumor carrier groups allow us to come to the conclusion that low-level irradiation has an impact only at the initial stages of the aftereffect. At the following stages, the state of the cytochrome part of the respiratory chain is defined by growth conditions of tumor.

Effect of laser on the remnant liver after the first 24 hours following 70 percent hepatectomy in rats / Efeito do laser no fígado remanescente nas primeiras 24 horas após hepatectomia a 70 por cento em ratos

PURPOSE: To evaluate the mitochondrial function of the remnant liver (RL) in the early phase of liver regeneration in rats after 70 percent partial hepatectomy (PH). METHODS: Sixty male Wistar rats (200-250g) submitted to 70 percent PH were divided into five groups according to the time of euthanasia and application or not of laser light: C = Control, time zero; 2 minutes, 4, 6 and 24 hours after PH. The dose of laser radiation was 22.5 J/cm², wavelength of 660 nm (visible/red), in the remnant liver. We studied the respiration activated by ADP (state 3), basal mitochondrial respiration (state 4), respiratory control ratio (RCR) and mitochondrial membrane potential (MMP). RESULTS: The mitochondrial function of RL changed at 4 and 6 hours after PH, with a significant increase in state 3 and a concomitant increase in state 4 and with maintenance of RCR. MMP differed significantly between the groups biostimulated with laser radiation and the control group 4 hours after HP, with a substantial reduction in the non-laser groups. CONCLUSION: The laser light at the dose used in this study did not induce additional damage to the RL and seems to have delayed the hepatocellular metabolic overload of the remnant liver.

OBJETIVO: Avaliar a função mitocondrial do fígado remanescente (FR) na fase precoce da regeneração hepática em ratos após hepatectomia parcial (HP) a 70 por cento. MÉTODOS: Sessenta ratos machos Wistar (200 - 250g) submetidos à HP a 70 por cento, foram distribuídos em cinco grupos de acordo com o tempo de eutanásia e com aplicação ou não de luz laser: C= Controle,tempo zero; 2 minutos, 4, 6 e 24 horas após HP. O laser foi utilizado na dose 22.5 J/cm², 660 nm, no FR.Estudou-se o estado 3 (respiração ativada por ADP), estado 4 (respiração mitocondrial basal), razão de controle respiratório,estado 3/estado 4 (RCR) e o potencial de membrana mitocondrial(PMM). RESULTADOS: A função mitocondrial do FR alterou-se no período de 4 e 6 horas após a HP com aumento significativo do estado 3 e aumento concomitante do estado 4, com manutenção da RCR. O PMM apresentou diferença significativa entre os grupos bioestimulados com laser e o controle a partir de 4 horas pós HP, com queda importante do grupo sem laser e tendência a equiparação dos valores após 24 horas. CONCLUSÃO: A luz laser, na dose utilizada no presente estudo, não induziu lesão adicional ao FR e parece ter retardado a sobrecarga hepatocelular do fígado remanescente.

Effect of laser on the remnant liver after the first 24 hours following 70% hepatectomy in rats.

PURPOSE: To evaluate the mitochondrial function of the remnant liver (RL) in the early phase of liver regeneration in rats after 70% partial hepatectomy (PH). METHODS: Sixty male Wistar rats (200-250g) submitted to 70% PH were divided into five groups according to the time of euthanasia and application or not of laser light: C = Control, time zero; 2 minutes, 4, 6 and 24 hours after PH. The dose of laser radiation was 22.5 J/cm(2), wavelength of 660 nm (visible/red), in the remnant liver. We studied the respiration activated by ADP (state 3), basal mitochondrial respiration (state 4), respiratory control ratio (RCR) and mitochondrial membrane potential (MMP). RESULTS: The mitochondrial function of RL changed at 4 and 6 hours after PH, with a significant increase in state 3 and a concomitant increase in state 4 and with maintenance of RCR. MMP differed significantly between the groups biostimulated with laser radiation and the control group 4 hours after HP, with a substantial reduction in the non-laser groups. CONCLUSION: The laser light at the dose used in this study did not induce additional damage to the RL and seems to have delayed the hepatocellular metabolic overload of the remnant liver.

Protective effect of an aminothiazole compound against γ-radiation induced oxidative damage.

Ionizing radiation causes its biological effects mainly through oxidative damage induced by reactive oxygen species. During radiotherapy of cancer, one of the undesirable side-effects is toxicity to normal cells. Compounds with antioxidant activities are being tried as 'prophylactic radioprotectants' to overcome this problem. We evaluated the protective effect of an aminothiazole compound, in the form of dendrodoine analogue (DA) originally derived from a marine tunicate, against γ-radiation-induced damage to lipid, protein, and DNA besides its cytotoxicity. Oxidative damage was examined by different biochemcial assays. Our studies reveal that DA gave significant protection, in fairly low concentrations, against damage induced by γ-radiation to rat liver mitochondria, plasmid pBR322 DNA, and mouse splenic lymphocytes in vitro. It also protected against oxidative damage in whole-body irradiated mice exposed to therapeutic dose of radiation (2 Gy) in vivo. Spleen, a major target organ for radiation damage, of the irradiated mice showed significant protection when treated with DA, as examined by histopathology. In conclusion, due to the possible protective effects against normal cells/tissues both in vitro and in vivo, DA shows potential to be a radioprotector for possible use during radiotherapy.

Neither nitrite nor nitric oxide mediate toxic effects of nitroglycerin on mitochondria.

It is commonly accepted that the major effect of nitroglycerin (NG) is realized through the release of nitric oxide (NO) catalyzed by aldehyde dehydrogenase-2 (ALDH2). In addition, it has been shown that NG inhibits mitochondrial respiration. The aim of this study was to clarify whether NG-mediated inhibition of mitochondrial respiration is mediated by NO. In rat liver mitochondria, NG inhibited complex-I-dependent respiration and induced reactive oxygen species (ROS) production, preferentially at complex I. Both effects were insensitive to chloral hydrate, an ALDH2 inhibitor. Nitrite, an NG intermediate, had no influence on either mitochondrial respiration or the production of ROS. NO inhibited preferentially complex I but did not elevate ROS production. Hemoglobin, an NO scavenger, and blue light had contrary effects on mitochondria inhibited by NO or NG. In summary, our data suggest that although NG induces vasodilatation via NO release, it causes mitochondrial dysfunction via an NO-independent pathway.

Mitophagy selectively degrades individual damaged mitochondria after photoirradiation.

Damaged and dysfunctional mitochondria are proposed to be removed by autophagy. However, selective degradation of damaged mitochondria by autophagy (mitophagy) has yet to be experimentally verified. In this study, we investigated the cellular fate of individual mitochondria damaged by photoirradiation in hepatocytes isolated from transgenic mice expressing green fluorescent protein fused to microtubule-associated protein 1 light chain 3, a marker of forming and newly formed autophagosomes. Photoirradiation with 488-nm light induced mitochondrial depolarization (release of tetramethylrhodamine methylester [TMRM]) in a dose-dependent fashion. At lower doses of light, mitochondria depolarized transiently with re-polarization within 3 min. With greater light, mitochondrial depolarization became irreversible. Irreversible, but not reversible, photodamage induced autophagosome formation after 32±5 min. Photodamage-induced mitophagy was independent of TMRM, as photodamage also induced mitophagy in the absence of TMRM. Photoirradiation with 543-nm light did not induce mitophagy. As revealed by uptake of LysoTracker Red, mitochondria weakly acidified after photodamage before a much stronger acidification after autophagosome formation. Photodamage-induced mitophagy was not blocked by phosphatidylinositol 3-kinase inhibition with 3-methyladenine (10 mM) or wortmannin (100 nM). In conclusion, individual damaged mitochondria become selectively degraded by mitophagy, but photodamage-induced mitophagic sequestration occurs independently of the phosphatidylinositol 3-kinase signaling pathway, the classical upstream signaling pathway of nutrient deprivation-induced autophagy.