Mitochondrial Radical Production Research Articles

Free radical quenching in liver mitochondria by selected antioxidants abundant in foods and supplements

The effects of several well-known natural and synthetic antioxidants on free radical production and oxygen uptake in rat liver mitochondria were measured. Ample concentration ranges (10−7 to 10−3 M) were explored in the search for stimulatory or inhibitory effects on both free radical production and oxygen uptake. The selected antioxidants are frequently present in foods, therapeutic formulations and supplements. Of the sixteen antioxidants, six of them increased free radical production in mitochondria at least over a certain concentration range, namely ascorbic acid, BHA, BHT, vanillic acid, caffeic acid and gallic acid. The last three inhibited free radical generation in the high concentration range. Several of the selected antioxidants behaved as inhibitors of energy metabolism at the concentrations causing 50% diminution of free radical production. The best inhibitors of free radical production in mitochondria were: resveratrol ≈ chlorogenic acid > epicatechin > rutin > ferulic acid. No significant correlations were found between the antioxidant strength in mitochondria and chemical systems nor was there any correlation between the antioxidant strength and the number of aromatic hydroxyls. Quantification of the free radical scavenging activity of a given compound or extract in chemical systems only exceptionally allows to infer its strength in mitochondria. It is proposed that the mitochondrial antioxidant assay should be used in addition to chemical assays when testing antioxidant preparations.

Preserving muscle health and wellbeing for long-term cancer survivors.

The anti-tumour activities of anthracycline antibiotics like doxorubicin (DOX) were first established in the 1960s. Since then they rank among the most effective anti-tumour therapeutics. An example of their impact is seen in the shift of 5 year survival rates for childhood cancer, from 30% to 80% since the 1960s (Stěrba et al. 2013). Topoisomerase IIα is a main target of anthracycline anti-tumour activity. Anthracyclines prevent topoisomerase-mediated relaxation of supercoiled DNA and subsequent DNA replication. Along with effectiveness as a cancer chemotherapy come dangerous side effects that include cardiovascular and respiratory muscle toxicity (Gilliam & St Clair, 2011, Stěrba et al. 2013). Toxicity can develop shortly after treatment or decades later, as is seen among childhood cancer survivors (Stěrba et al. 2013). The mechanism of anthracycline-induced muscle toxicity is still in question. Contributing factors include oxidative stress, calcium dysregulation, and calpain degradation of titin (Stěrba et al. 2013). Muscle mitochondria appear to be at the centre of the problem. First, anthracyclines, known for their ability to produce free radicals, concentrate in mitochondria via an affinity for cardiolipin (Stěrba et al. 2013). Cardiolipin is an inner mitochondrial membrane phospholipid associated with cytochrome c (Kroemer et al. 2007). Second, mitochondria subjected to oxidative stress affect cellular calcium dynamics (Kroemer et al. 2007). Thus, concentrated anthracycline-mediated free radical production in mitochondria might alter cellular calcium dynamics, and subsequent calcium-regulated calpain activity. In this issue of The Journal of Physiology, Min et al. (2015) linked DOX-induced mitochondrial oxidative stress to calpain activation and cardiac/skeletal muscle dysfunction. They demonstrated that pretreatment with the mitochondrial specific antioxidant SS-31, or the calpain inhibitor SJA 6017, protected rats from DOX-induced muscle toxicity. SS-31 preserved mitochondrial respiratory function, reduced cellular oxidative stress, and prevented calpain activation. Both SS-31 and SJA preserved diastolic and systolic cardiac function and diaphragm contractile function, and reduced skeletal muscle atrophy. The fact that SS-31 prevented calpain activation in heart and skeletal muscle supports the idea that DOX alters mitochondrial redox balance to stimulate calpain (Fig.​(Fig.1).1). Interestingly, both DOX and SS-31 target mitochondria through interaction with cardiolipin (Szeto, 2014). The key to SS-31 efficacy may lie in this interaction. As mentioned above, cardiolipin associates with a major fraction of mitochondrial cytochrome c and cardiolipin oxidation leads to cytochrome c release (Orrenius & Zhivotovsky, 2005). Thus, disruption of cardiolipin–cytochrome c complexes either by physical interaction with, or oxidation by, DOX could lead to cytochrome c release. The resulting cytosolic cytochrome c would promote Ca2+ release from the endoplasmic reticulum (Orrenius & Zhivotovsky, 2005) to activate calpain (Kroemer et al. 2007). SS-31 disruption of a DOX-cardiolipin interaction or cardiolipin oxidation could prevent the initiation of this cytotoxic cascade. Figure 1 Mitochondria and muscle toxicity In summary, these results provide further evidence that mitochondria are central to anthracycline-induced muscle toxicity. They also define a pathway initiated by DOX that triggers mitochondrial changes which stimulate calpain to promote muscle dysfunction and atrophy. Finally they demonstrate the potential for therapies targeted to mitochondria or calpain that would preserve muscle function and prevent dangerous long-term side effects of cancer chemotherapy.

Sites of reactive oxygen species generation by mitochondria oxidizing different substrates

Mitochondrial radical production is important in redox signaling, aging and disease, but the relative contributions of different production sites are poorly understood. We analyzed the rates of superoxide/H2O2 production from different defined sites in rat skeletal muscle mitochondria oxidizing a variety of conventional substrates in the absence of added inhibitors: succinate; glycerol 3-phosphate; palmitoylcarnitine plus carnitine; or glutamate plus malate. In all cases, the sum of the estimated rates accounted fully for the measured overall rates. There were two striking results. First, the overall rates differed by an order of magnitude between substrates. Second, the relative contribution of each site was very different with different substrates. During succinate oxidation, most of the superoxide production was from the site of quinone reduction in complex I (site IQ), with small contributions from the flavin site in complex I (site IF) and the quinol oxidation site in complex III (site IIIQo). However, with glutamate plus malate as substrate, site IQ made little or no contribution, and production was shared between site IF, site IIIQo and 2-oxoglutarate dehydrogenase. With palmitoylcarnitine as substrate, the flavin site in complex II (site IIF) was a major contributor (together with sites IF and IIIQo), and with glycerol 3-phosphate as substrate, five different sites all contributed, including glycerol 3-phosphate dehydrogenase. Thus, the relative and absolute contributions of specific sites to the production of reactive oxygen species in isolated mitochondria depend very strongly on the substrates being oxidized, and the same is likely true in cells and in vivo.

The Functional and Neuroprotective Actions of Neu2000, a Dual-Acting Pharmacological Agent, in the Treatment of Acute Spinal Cord Injury

The goal of the present study was to examine the neuroprotective and functional significance of targeting both N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity and oxidative stress using a dual-acting compound, Neu2000, in rat model of moderate spinal cord injury (SCI). An initial set of experiments was conducted in uninjured rats to study the pharmacokinetic profile of Neu2000 following intraperitoneal and intravenous administration. A second experiment measured free radical production in mitochondria isolated from sham or injured spinal cords of animals receiving vehicle or Neu2000 treatment. A third set of animals was divided into three treatment groups consisting of vehicle treatment, a single dose of Neu2000 (50 mg/kg) administered at 10 min following injury, or a repeated treatment paradigm consisting of a single bolus of Neu2000 at 10 min following injury (50 mg/kg) plus a maintenance dose (25 mg/kg) administered every 24 h for an additional 6 days. Animals were tested once a week for a period of 6 weeks for evidence of locomotor recovery in an open field and kinematic analysis of fine motor control using the DigiGait Image Analysis System. At the end of the testing period, spinal cord reconstruction was performed to obtain nonbiased stereological measures of tissue sparing. The results of this study demonstrate that Neu2000 treatment significantly reduced the production of mitochondrial free radicals and improved locomotor outcomes that were associated with a significant increase in the volume of spared spinal cord tissue.

2-chlorophenol induced hydroxyl radical production in mitochondria in Carassius auratus and oxidative stress – An electron paramagnetic resonance study

In our previous study, electron paramagnetic resonance (EPR) evidence of reactive oxygen species (ROS) production in Carassius auratus following 2-chlorophenol (2-CP) administration was provided. To further investigate the potential pathway of ROS production, liver mitochondria of C. auratus was isolated and incubated with 2-CP for 30min. An EPR analysis indicated ROS was produced, and intensities of ROS increased with increasing concentrations of 2-CP. The ROS was then assigned •OH by comparing with Fenton reaction. Either catalase or superoxide dismutase, extinguished •OH completely in the mitochondria mixture. These facts suggested that O2·- and H2O2 contributed to the formation of •OH in mitochondria in C. auratus stressed by 2-CP. Combining previous references and our own data, it is reasonable to suggest that 2-CP is first oxidized by H2O2 present in vivo to form phenoxyl radical under the catalytic action of cellular peroxidase (1); phenoxyl radical oxidizes mitochondria NADH to NAD• in the presence of NADH (2); NAD• reacts with oxygen in vivo to produce O2·- (3); O2·- is spontaneously dismutated by SOD to form H2O2 and O2, which creates a renewable supply of H2O2 as the initiators of the chain reactions until NADH is consumed (4); simultaneously with reaction (4), O2·- reacts with H2O2 to form •OH radical via the Haber–Weiss reaction (5). A strong negative correlation (r=−0.9278, p<0.01) between glutathione (GSH) pool and •OH production was observed after fish were i.p. injected with 2-CP (250mgkg−1), indicating the depletion of GSH caused by •OH.

A carboxyfullerene SOD mimetic improves cognition and extends the lifespan of mice

In lower organisms, such as Caenorhabditis elegans and Drosophila, many genes identified as key regulators of aging are involved in either detoxification of reactive oxygen species or the cellular response to oxidatively-damaged macromolecules. Transgenic mice have been generated to study these genes in mammalian aging, but have not in general exhibited the expected lifespan extension or beneficial behavioral effects, possibly reflecting compensatory changes during development. We administered a small-molecule synthetic enzyme superoxide dismutase (SOD) mimetic to wild-type ( i.e. non-transgenic, non-senescence accelerated) mice starting at middle age. Chronic treatment not only reduced age-associated oxidative stress and mitochondrial radical production, but significantly extended lifespan. Treated mice also exhibited improved performance on the Morris water maze learning and memory task. This is to our knowledge the first demonstration that an administered antioxidant with mitochondrial activity and nervous system penetration not only increases lifespan, but rescues age-related cognitive impairment in mammals. SOD mimetics with such characteristics may provide unique complements to genetic strategies to study the contribution of oxidative processes to nervous system aging.

Na-K-ATPase in rat cerebellar granule cells is redox sensitive

Redox-induced regulation of the Na-K-ATPase was studied in dispersed rat cerebellar granule cells. Intracellular thiol redox state was modulated using glutathione (GSH)-conjugating agents and membrane-permeable ethyl ester of GSH (et-GSH) and Na-K-ATPase transport and hydrolytic activity monitored as a function of intracellular reduced thiol concentration. Depletion of cytosolic and mitochondrial GSH pools caused an increase in free radical production in mitochondria and rapid ATP deprivation with a subsequent decrease in transport but not hydrolytic activity of the Na-K-ATPase. Selective conjugation of cytosolic GSH did not affect free radical production and Na-K-ATPase function. Unexpectedly, overloading of cerebellar granule cells with GSH triggered global free radical burst originating most probably from GSH autooxidation. The latter was not followed by ATP depletion but resulted in suppression of active K(+) influx and a modest increase in mortality. Suppression of transport activity of the Na-K-ATPase was observed in granule cells exposed to both permeable et-GSH and impermeable GSH, with inhibitory effects of external and cytosolic GSH being additive. The obtained data indicate that redox state is a potent regulator of the Na-K-ATPase function. Shifts from an "optimal redox potential range" to higher or lower levels cause suppression of the Na-K pump activity.

The efficiency and plasticity of mitochondrial energy transduction

Since it was first realized that biological energy transduction involves oxygen and ATP, opinions about the amount of ATP made per oxygen consumed have continually evolved. The coupling efficiency is crucial because it constrains mechanistic models of the electron-transport chain and ATP synthase, and underpins the physiology and ecology of how organisms prosper in a thermodynamically hostile environment. Mechanistically, we have a good model of proton pumping by complex III of the electron-transport chain and a reasonable understanding of complex IV and the ATP synthase, but remain ignorant about complex I. Energy transduction is plastic: coupling efficiency can vary. Whether this occurs physiologically by molecular slipping in the proton pumps remains controversial. However, the membrane clearly leaks protons, decreasing the energy funnelled into ATP synthesis. Up to 20% of the basal metabolic rate may be used to drive this basal leak. In addition, UCP1 (uncoupling protein 1) is used in specialized tissues to uncouple oxidative phosphorylation, causing adaptive thermogenesis. Other UCPs can also uncouple, but are tightly regulated; they may function to decrease coupling efficiency and so attenuate mitochondrial radical production. UCPs may also integrate inputs from different fuels in pancreatic beta-cells and modulate insulin secretion. They are exciting potential targets for treatment of obesity, cachexia, aging and diabetes.

Radical mediators and mitogen-activated protein kinase signaling in oxygen-dependent radiosensitivity of human tumor cell lines

Oxygen enhancement of tumor radiosensitivity is attributed to DNA damage by reactive oxygen species. The mechanism remains unclear but may involve mitochondria as major sources of oxygen and nitrogen radicals as well as central effectors of energy homeostasis and apoptosis. Here we used dihydrorhodamine and 2′,7′-dichlorodihydrofluorescein to compare mitochondrial and total cell generation, respectively, of reactive oxygen or nitrogen species in cells irradiated at 5 Gy. Irradiation in the presence of oxygen selectively stimulated mitochondrial radical production in HeLa and MeWo cells, but in MCF7 cells radical production was more generalized. In all three cell lines oxygen impaired cell proliferation as measured by resazurin reduction 7 days after irradiation. Antioxidants N-acetylcysteine, ascorbic acid, and melatonin largely prevented dye oxidation during normoxic irradiation yet had no effect on oxygen-dependent irradiation injury. However, NO synthase inhibitor N( G)-monomethyl- l-arginine protected HeLa and MCF7 though not MeWo cells, consistent with their different levels of constitutive NO generation. SB203580 inhibition of p38 MAPK appreciably protected HeLa and marginally protected MCF7 cells against oxygen-dependent irradiation injury, while the less specific JNK/SAPK inhibitor SP600125 and ERK inhibitor U0126 had no effect. None of the inhibitors affected MeWo radiosensitivity. Therefore oxygen-enhanced radiosensitivity in these tumor cell lines does not depend on extensive production of oxygen radicals and is cell-type dependent. NO mediates oxygen-dependent injury in HeLa and MCF7 cells, by p38-dependent and MAPK–independent mechanisms, respectively. In MeWo cells this oxygen-enhanced radiosensitivity is independent of both NO and MAPK signaling.

Investigating mitochondrial radical production using targeted probes

A range of mitochondria-targeted probe molecules that comprise a lipophilic cation covalently attached to an active moiety have been developed. The lipophilic cation causes the accumulation of these molecules into mitochondria, driven by the mitochondrial membrane potential. To date, we have targeted antioxidants, spin traps, thiol reagents and DNA-alkylating compounds to mitochondria. The selective mitochondrial localization of these compounds enables us to investigate several aspects of the production of reactive oxygen species by mitochondria.

Prevention of mitochondrial oxidative damage as a therapeutic strategy in diabetes.

Hyperglycemia causes many of the pathological consequences of both type 1 and type 2 diabetes. Much of this damage is suggested to be a consequence of elevated production of reactive oxygen species by the mitochondrial respiratory chain during hyperglycemia. Mitochondrial radical production associated with hyperglycemia will also disrupt glucose-stimulated insulin secretion by pancreatic beta-cells, because pancreatic beta-cells are particularly susceptible to oxidative damage. Therefore, mitochondrial radical production in response to hyperglycemia contributes to both the progression and pathological complications of diabetes. Consequently, strategies to decrease mitochondrial radical production and oxidative damage may have therapeutic potential. This could be achieved by the use of antioxidants or by decreasing the mitochondrial membrane potential. Here, we outline the background to these strategies and discuss how antioxidants targeted to mitochondria, or selective mitochondrial uncoupling, may be potential therapies for diabetes.

Using mitochondria-targeted molecules to study mitochondrial radical production and its consequences.

The production of ROS (reactive oxygen species) by the mitochondrial respiratory chain contributes to a range of pathologies, including neurodegenerative diseases, ischaemia/reperfusion injury and aging. There are also indications that mitochondrial ROS production plays a role in damage response and signal transduction pathways. To unravel the role of mitochondrial ROS production in these processes, we have developed a range of mitochondria-targeted probe molecules. Covalent attachment of a lipophilic cation leads to their accumulation into mitochondria, driven by the membrane potential. Molecules developed so far include antioxidants designed to intercept mitochondrial ROS and reagents that specifically label mitochondrial thiol proteins. Here we outline how mitochondrial ROS formation and its consequences can be investigated using these probes.

Measurements of protein carbonyls, ortho- and meta-tyrosine and oxidative phosphorylation complex activity in mitochondria from young and old rats

Mitochondrial bioenergetic function is often reported to decline with age and the accumulation of oxidative damage is thought to contribute. However, there are considerable uncertainties about the amount and significance of mitochondrial oxidative damage in aging. We hypothesized that, as radical production in mitochondria is greater than the rest of the cell, protein oxidative damage should accumulate more in mitochondria than the cytoplasm, and that this relative accumulation should increase with age. To test these hypotheses we measured the accumulation of three markers of protein oxidative damage in liver, brain, and heart from young and old rats. Ortho- and meta-tyrosine levels in protein hydrolysates were measured by a gas chromatography/mass spectrometry assay, and protein carbonyl content was determined by ELISA. Using these assays we found no evidence for increased protein oxidative damage in mitochondria relative to the cytosol. Most increases found in protein oxidative damage on aging were modest for all three tissues and there was no consistent pattern of increased oxidative damage in mitochondrial proteins on aging. Mitochondrial oxidative phosphorylation complex activities were also assessed revealing 39–42% decreases in F 0F 1 - ATP synthase activity in liver and heart on aging, but not in other oxidative phosphorylation complexes. These findings have implications for the contribution of mitochondrial oxidative damage and dysfunction to aging.

Role of energy metabolism in the preconditioned heart--a possible contribution of mitochondria.

A brief period of ischemia and reperfusion has been shown to protect the myocardium against subsequent sustained ischemia and reperfusion injury, which is called "preconditioning". A great number of investigators have explored the mechanisms underlying this preconditioning-induced cardioprotection. This article dealt with possible mechanisms of energy metabolism and mitochondrial activity for preconditioning-induced cardioprotection. Particularly, the contribution of energy metabolites produced during a brief period of ischemia and reperfusion injury, as well as mitochondrial function that is modified by changes in mitochondrial ATPase activity, opening of mitochondrial ATP-dependent potassium channels and production of free radicals in mitochondria, to ischemic preconditioning is discussed.

Energy Metabolism, Nutrition and Ageing

Abstract. Energy metabolism and nutrition have long been suggested to play a role in ageing processes, in age‐related disease and in the health of the elderly. This review addresses new insights arising from selected studies of the interplay of these factors. Data on overfeeding and underfeeding in young versus elderly men and women demonstrate a deficit with age in the ability to regulate energy balance. They also indicate that current values of Recommended Daily Allowances of caloric intake underestimate the needs of the elderly. The studies suggest that diminished capacity of energy metabolism with age may have a nutritional component and the involvement of the central nervous system. The contribution of peripheral tissues is suggested by studies of mitochondrial DNA isolated from tissues of humans and rodents. The studies demonstrate age‐related and disease‐related increases in rates of mutation in some but not all tissues. The further involvement of mitochondrial dysfunction in ageing is suggested by data on oxidative free radical production in mitochondria of birds versus rodents. These elegant studies indicate greatly reduced rates of free radical production in mitochondria of long‐lived ‐ birds versus short‐lived rodents. They further demonstrate that Complex I of the respiratory chain may be a major factor in these species‐specific effects. Restriction of caloric intake (dietary restriction, DR) is the only manipulation known to consistently extend lifespan in a wide variety of species. Previous work shows that extension of lifespan in DR is not necessarily related to reduced metabolic rate per unit metabolic mass. More recent work in exercising DR rats demonstrates consistently high daily metabolic rates together with extended lifespan. Other studies also show altered characteristics of fuel use in DR, indicating the ability of DR rats to maintain appropriate rates of fuel use under conditions less damaging to the organism. All of these new approaches to the interaction of nutrition, energy expenditure and ageing suggest that regulation of energy balance and the characteristics of fuel use play significant roles in ageing and in age‐related disease.

Enhanced mitochondrial radical production in patients which rheumatoid arthritis correlates with elevated levels of tumor necrosis factor alpha in plasma.

Mitochondrial dysfunction contributes to cell damage in a number of human diseases. One significant mechanism by which mitochondria damage cells is by producing reactive oxygen species from the respiratory chain. In this study we measured the production of reactive oxygen species by leukocyte mitochondria in blood from rheumatoid arthritis patients. To do this we used the chemiluminescence of lucigenin, which is accumulated by mitochondria within cells and reacts with superoxide to form a chemiluminescent product. By using specific inhibitors we could distinguish between the production of reactive oxygen species by mitochondria and by NADPH oxidase. There was a five-fold increase in mitochondrial reactive oxygen species production in whole blood and monocytes from patients with rheumatoid arthritis, when compared to healthy subjects or patients with non-rheumatic diseases. There was no increases in mitochondrial reactive oxygen species production by neutrophils from rheumatoid arthritis patients. The enhanced mitochondrial radical production in rheumatoid arthritis patients correlated significantly with increased levels of tumor necrosis factor alpha in plasma (p < 0.0001). As tumor necrosis factor alpha is known to increase mitochondrial reactive oxygen species production the elevated mitochondrial radical formation seen in rheumatoid arthritis patients may be due to activation of the mitochondrial radical production. These data suggest that elevated mitochondrial oxidative stress contributes to the pathology of rheumatoid arthritis.

Isolated Cerebral and Cerebellar Mitochondria Produce Free Radicals when Exposed to Elevated Ca2+ and Na+: Implications for Neurodegeneration

The evidence is compelling that free radicals, plus increases in free cytosolic Ca2+ and Na+, figure prominently in neuronal death after exposure to glutamate and dicarboxylic excitotoxins such as NMDA and kainate. However, neither the source of these radicals nor the direct connection between Ca2+ mobilization and radical production has been well defined. Electron paramagnetic resonance studies reported here indicate that intact mitochondria isolated from adult rat cerebral cortex and cerebellum generate extremely reactive hydroxyl (.OH) radicals, plus ascorbyl and other carbon-centered radicals when exposed to 2.5 microM Ca2+, 14 mM Na+, plus elevated ADP under normoxic conditions, circumstances that prevail in the cytoplasm of neurons during excitotoxin-induced neurodegeneration. In a feed-forward cycle, exposure of isolated mitochondria to .OH significantly increases subsequent radical production five- to 16-fold (average = 8.8 +/- 1.6 SE, n = 6, p > 0.01) with succinate as substrate, and also selectively impairs function of NADH-CoQ dehydrogenase activity (electron transport complex 1). These effects are also reflected by respiration rates that are reduced 48% with complex 1 substrates, but increased 27% with complex 2 substrate, after .OH exposure. Comparable complex 1 dysfunction is observed in mitochondria isolated from the substantia nigra of Parkinson's disease patients, from platelets of Huntington's disease patients, and from neocortex of Alzheimer's disease patients. Mitochondrial radical production provides a testable model, based on oxyradical toxicity, oxidative enzyme inactivation, and mitochondrial dysfunction, for the final common pathway of neuronal necrosis during excitotoxicity, and in a host of neurodegenerative disorders.

Radical production in liver mitochondria by peroxidic tumor promoters

Eleven peroxides have been tested to determine if there is a correlation between tumor-promoting activity and the ability to stimulate radical production in mitochondria. When non-respiring rat liver mitochondria are treated with these peroxidic compounds in the presence of DMPO, ESR signals are observed from the spin trapping of carbon- and oxygen-centered radicals in the case of 4 of the 7 peroxides that are known to be tumor promoters. Enhancement of carbon-centered radical production is observed in the presence of respiratory substrate. Thus there does not appear to be a correlation between tumor-promoting activity of peroxidic compounds and radical production in mitochondria. Oxidants can act as promoters either by 1- or 2-electron oxidation pathways; both types of mechanisms may be inhibited by antioxidants, which can scavenge either radicals or electrophiles.

Oxygen detoxification in algal-invertebrate symbioses from the Great Barrier Reef.

Examination of 34 species of symbiotic invertebrates in four phyla has confirmed the generality of a direct relationship between chlorophyll concentration and the activities of superoxide dismutase and catalase, two enzymes involved in the detoxification of active oxygen. On a finer scale, activities of these enzymes also depend on the localization of the algal symbions (intracellular or extracellular) and hence on the extent to which photosynthetic O2 actually contacts animal cytoplasm, and on the solar irradiance experienced by the symbionts. Differences in SOD activity among organs of Tridacna crocea are not fully explained by local O2 levels but are further related to organ-specific retes of O2 consumption. This result is discussed in terms of known mechanisms of superoxide radical production in mitochondria and differences in O2 utilization concentrations among various organs in bivalve molluscs.