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Dependence of H2O2 Formation by Rat Heart Mitochondria on Substrate Availability and Donor Age
Abstract
We have examined the substrate specificity and inhibitor sensitivity of H2O2 formation by rat heart mitochondria. Active H2O2 production requires both a high fractional reduction of Complex I (indexed by NADH/NAD+ + NADH ratio) and a high membrane potential, delta psi. These conditions are achieved with supraphysiological concentrations of succinate. With physiological concentrations of NAD-linked substrates, rates of H2O2 formation are much lower (less than 0.1% of respiratory chain electron flux) but may be stimulated by the Complex III inhibitor antimycin A, but not by myxothiazol. Addition of Mn2+ to give 10 nmol/mg of mitochondrial protein enhances H2O2 production with all substrate combinations, possibly by repleting mitochondrial superoxide dismutase with this cation. Contrary to previously published work, no increased activity of H2O2 production was found with heart mitochondria from senescent (24 month) rats, relative to young adults (6 month).
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“Δψm is an important factor for maintaining cellular energy homeostasis, and small reductions in Δψm induced by mild uncoupling were shown to decrease H2O2 emissions (51,52) and produce a natural antioxidant effect (53).”
From TFAM Enhances Fat Oxidation and Attenuates High-Fat Diet–Induced Insulin Resistance in Skeletal Muscle (2019) · cites this paper as [51]
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“RET and the related NADH generation in succinate-supported mitochondria are prevented by the complex I inhibitor rotenone [20,32,42,90], but this did not alter the MBevoked stimulation of respiration (Table 1).”
InfluentialFeidantsis, K. G., Giantsis, I. Α., Vratsistas, A., Makri, S., Pappa, A.-Z., Drosopoulou, E., Anestis, A., Mavridou, E., Exadactylos, A., Vafidis, D., et al. (2020) · American Journal of Physiology-Regulatory, Integrative and Comparative Physiology · doi:10.1152/ajpregu.00066.2020 · Cited by 65
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“The level of superoxide formed under physiologic conditions, when mitochondria are respiring primarily on NAD-linked substrates, has been estimated to account for 0.1% to 0.4% of electron flow (23, 65).”
InfluentialKhailova, L. S., Silachev, D. N., Rokitskaya, T. I., Avetisyan, A. V., Lyamsaev, K. G., Severina, I. I., Ilyasova, T. M., Gulyaev, M. V., Dedukhova, V. I., Trendeleva, T. A., et al. (2014) · Biochimica et Biophysica Acta (BBA) - Bioenergetics · doi:10.1016/j.bbabio.2014.07.006 · Cited by 62
“Even a small decrease in Δψ initially exceeding the level of approximately 150 mV was shown to disproportionately decrease H2O2 generation by mitochondria [6–9].”
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“1 confirm previous reports [14 – 16], showing mitochondrial ROS generation in the presence of succinate alone is largely inhibited by the Cx-I inhibitor rotenone (Fig.”
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“Myxothiazol has been documented to augment ROS release from cardiac mitochondria due to the over reduction of electron donating centers in Complex I [29,42].”
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“Indeed, superoxide production is reduced when reverse electron transfer is inhibited with rotenone (6, 44).”
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“This has been shown to be sensitive to inhibition of complex I by rotenone and related to a reverse electron flow through complex I (9,10,22,24).”
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Mitochondrial Toxin 3-Nitropropionic Acid Induces Cardiac and Neurotoxicity Differentially in Mice
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Self-citationDisturbance of redox homeostasis in Down Syndrome: Role of iron dysmetabolism
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“The major source of free radicals is the mitochondrial oxidative phosphorylation pathway, in which electron leakage from the electron transport chain causes the formation of O2 that, in turn, is converted by mitochondrial-resident MnSOD into H2O2 and O2 [25].”
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“These early estimates have been proven to be excessive, with more recent studies reporting values in the range of 0.15 to 1.0% (Hansford et al., 1997; Lopez-Torres et al., 2002; St-Pierre et al., 2002; Gomez et al., 2009), which agree with values we obtained for parrots and quails.”
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“In this present study we also found that E2 increased mitochondrial membrane potential, a fact that is strongly linked to ROS production [Hansford et al., 1997; Korshunov et al., 1997; Brand et al., 2004].”
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“Because RONS production is especially associated with dysfunction of complexes I and III [11–14], data were also analyzed excluding the three subjects who did not have a deficiency of either of these complexes.”
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Civitarese, A. E., Smith, S. R., & Ravussin, É. (2007) · Current Opinion in Clinical Nutrition & Metabolic Care · doi:10.1097/mco.0b013e3282f0ecd2 · Cited by 52
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Skeletal muscles, heart, and lung are the main sources of oxygen radicals in old rats
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“While some studies have shown that mitochondria produce more RONS with the age [4–9], others did not find any increase in mitochondrial RONS generation [10,11].”
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“It is estimated that 0.2 to 2% of the oxygen consumed by a cell is transformed to ROS by electron leakage during oxidative phosphorylation (Chance et al., 1979; Hansford et al., 1997).”
BackgroundInfluentialReactive oxygen species formation and its effect on CD4+ T cell-mediated inflammation
Shu, P., Liang, H., Zhang, J., Lin, Y., Chen, W., & Zhang, D. (2023) · Frontiers in Immunology · doi:10.3389/fimmu.2023.1199233 · Cited by 58
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“…force is sufficient to allow the flow of a fraction of electrons in reverse (reverse electron transfer; RET) producing NADH (Chance and Hollunger 1961a; Hinkle et al. 1967) and superoxide by complex I (Hansford et al. 1997; Korshunov et al. 1997; Votyakova and Reynolds 2001; Liu et al. 2002).”
BackgroundResultInfluentialCardiac mitochondrial complex activity is enhanced by heat shock proteins
Sammut, I. A., & Harrison, J. C. (2003) · Clinical and Experimental Pharmacology and Physiology · doi:10.1046/j.1440-1681.2003.03799.x · Cited by 50
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“Even under normoxia, a fraction of the reducing equivalents handled by mitochondrial metabolism escapes the respiratory chain as superoxide, most of which will be converted to hydrogen peroxide (Hansford et al. 1997).”
BackgroundRedox signaling and skeletal muscle adaptation during aerobic exercise
Zhou, Y., Zhang, X., Baker, J. S., Davison, G. W., & Yan, X. (2024) · iScience · doi:10.1016/j.isci.2024.109643 · Cited by 52
“24,25 During exercise, OXPHOS becomes more efficient, resulting in a decrease in O 2 $ - production.”
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Increase in mitochondrial biogenesis, oxidative stress, and glycolysis in murine lymphomas
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“After the cells were washed in PBS with 1% BSA, cellular levels of ROS and mitochondria were determined by flow cytometry in a Becton–Dickinson LSR cytometer, and data were analyzed using the WinMDI version 2.8 software package.”
BackgroundInfluentialWong, H. S., Monternier, P., & Brand, M. D. (2019) · Free Radical Biology and Medicine · doi:10.1016/j.freeradbiomed.2019.09.006 · Cited by 46
Metabolism, ubiquinone synthesis, and longevityPreprint
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“4% to 4% of molecular oxygen is only partially reduced, producing the superoxide ion (O2 ) (Boveris and Chance 1973; Turrens and Boveris 1980; Boveris 1984; Imlay and Fridovich 1991; Hansford et al. 1997).”
BackgroundInfluentialLeman, G., Pavel, P., Hermann, M., Crumrine, D. A., Elias, P. M., Minzaghi, D., Goudounèche, D., Prieto, N. M. R., Cavinato, M. S., Wanner, A., et al. (2022) · Journal of Investigative Dermatology · doi:10.1016/j.jid.2022.01.035 · Cited by 46
Mitochondrial superoxide flashes: From discovery to new controversies
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“…activated by) bursts in superoxide produced by the ETC. Results from isolated mitochondria demonstrate a very steep nonlinear relationship between the mitochondrial membrane potential and ROS produced by complex I (Hansford et al., 1997; Votyakova and Reynolds, 2001; Miwa and Brand, 2003).”
Physical training prevents oxidative stress in L‐NAME‐induced hypertension rats
Cardoso, A. M., Martins, C. C., Fiorin, F. D. S., Schmatz, R., Abdalla, F. H., Gutierres, J. M., Zanini, D., Fiorenza, A. M., Stefanello, N., Serres, J. D. D. S., et al. (2012) · Cell Biochemistry and Function · doi:10.1002/cbf.2868 · Cited by 43
“Furthermore, Hansford et al.(71) found an increase on H2O2 production by rat heart mitochondria with lower values of O2 in the respiratory chain and using inhibitors, which can be similar to what happens in an exercise session.”
BackgroundOelkrug, R., Goetze, N., Meyer, C. W., & Jastroch, M. (2014) · Free Radical Biology and Medicine · doi:10.1016/j.freeradbiomed.2014.09.004 · Cited by 41
“It is established that high MMP causes increased electron leakage, thus increasing ROS production [1,23–26].”
BackgroundStimulation of H2O2 generation by calcium in brain mitochondria respiring on α‐glycerophosphate
Tretter, L., Takacs, K., Kövér, K., & Ádám‐Vizi, V. (2007) · Journal of Neuroscience Research · doi:10.1002/jnr.21405 · Cited by 42
“, 1967), leading to NADH formation (Hansford et al., 1997; Liu et al., 2002; Gyulkhandanyan and Pennefather, 2004) and ROS generation by Complex I (Boveris and Chance, 1973; Korshunov et al.”
BackgroundInfluentialSilachev, D. N., Khailova, L. S., Babenko, V. A., Gulyaev, M. V., Kovalchuk, S. I., Zorova, L. D., Plotnikov, E. Y., Antonenko, Y. N., & Zorov, D. B. (2014) · Biochimica et Biophysica Acta (BBA) - General Subjects · doi:10.1016/j.bbagen.2014.09.002 · Cited by 41
“Due to this, high values of the membrane potential may be deleterious, specifically under pathological conditions associated with oxidative stress [4,5].”
BackgroundCytotoxicity of Ferulic Acid on T24 Cell Line Differentiated by Different Microenvironments
Peng, C.-C., Chyau, C.-C., Wang, H.-E., Chang, C.-H., Chen, K., Chou, K., & Peng, R. Y. (2013) · BioMed Research International · doi:10.1155/2013/579859 · Cited by 41
Effect of aging on formation of reactive oxygen species by mitochondria of rat heart
Kuka, S., Tatarková, Z., Račay, P., Lehotský, J., Dobrota, D., & Kaplán, P. (2014) · General Physiology and Biophysics · doi:10.4149/gpb_2013049 · Cited by 43
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“ROS production with succinate is mainly generated by reverse electron flux from complex II to complex I [62 – 65] and is at least threefold more sensitive to the pH gradient ( Δ pH) across the mitochondrial inner membrane than to the membrane potential ( Δ ψ ) [43].”
Aguirre, E., & Cadenas, S. (2010) · Biochimica et Biophysica Acta (BBA) - Bioenergetics · doi:10.1016/j.bbabio.2010.06.009 · Cited by 42
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“2–2% of O2 taken up by cells is converted to reactive O2 species, through mitochondrial superoxide generation [52].”
BackgroundDissociation of superoxide production by mitochondrial complex I from NAD(P)H redox state
Lambert, A. J., Buckingham, J. A., & Brand, M. D. (2008) · FEBS Letters · doi:10.1016/j.febslet.2008.04.030 · Cited by 40
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“This provides the most likely explanation for the DYm-dependent nature of ROS formation observed in succinatesupported mitochondria [16, 17, 20, 23, 32].”
BackgroundResultInfluentialExogenous insulin can reverse the effects of caloric restriction on mitochondria
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Metadata
- Authors
- Hansford, R. G., Hogue, B. A., & Mildaziene, V.
- Affiliations
- National Institute on Aging (US)
- Venue
- Journal of Bioenergetics and Biomembranes
- Published
- 1997-02
- Volume
- 29
- Issue
- 1
- Pages
- 89-95
- ISSN
- 0145-479X (ISSN-L)
- 1573-6881
- OpenAlex
- W2414123687
- Cited by
- 447
- References
- 26
- Citations by year
- 2026: 4 · 2025: 7 · 2024: 9 · 2023: 2 · 2022: 12 · 2021: 11 · 2020: 10 · 2019: 15 · 2018: 13 · 2017: 11 · 2016: 11 · 2015: 11 · 2014: 26 · 2013: 17 · 2012: 28
- FWCI
- 5.10
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- Locations
- Journal of Bioenergetics and Biomembranes (publishedVersion)
- PubMed (publishedVersion)
- Lithuanian University of Health Sciences (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
- Health Sciences › Medicine › Physiology (medical) › Nitric Oxide and Endothelin Effects
- Keywords
- hydrogen peroxide production, heart mitochondria, mitochondrial reactive oxygen species, complex I, NAD+/NADH ratio, membrane potential, succinic acid, antimycin A, myxothiazol, manganese, superoxide dismutase, donor age, senescence, substrate specificity, inhibitor sensitivity
- MeSH terms
- Aging, Animals, Electron Transport, Hydrogen Peroxide, Mitochondria, Heart, Substrate Specificity, Succinates, Uncoupling Agents, Rats
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