Respiratory Activity and Naphthoquinone Synthesis in the Fungus Fusarium decemcellulare Exposed to Oxidative Stress

The effect of oxidants (hydrogen peroxide and juglone) on the growth, respiration, and naphthoquinone synthesis in the fungus Fusarium decemcellulare was studied. The addition of the oxidants to the exponential-phase fungus inhibited cell respiration (either partially or completely, depending on the oxidant concentration), culture growth, and naphthoquinone synthesis. The treatment of fungal cells with nonlethal concentrations of H₂O₂ (below 0.25 mM) and juglone (below 0.1 mM) induced the resistance of cell respiration to cyanide. The residual respiration in the presence of cyanide could be inhibited by benzohydroxamic acid, indicating the occurrence of alternative oxidase. Increased concentrations of oxidants (0.25 mM juglone and 0.5 mM H₂O₂) rapidly and irreversibly inhibited cell respiration. These observations suggest that the mitochondrial respiratory chain of fungal cells exposed to oxidative stress is subject to the action of active oxygen species. The treatment of fungal cells with nonlethal concentrations of H₂O₂ and juglone activated cellular glutathione reductase and glucose-6-phosphate dehydrogenase, which are protective enzymes against oxidative stress.     

Adaptation of the phytopathogenic fungus Fusarium decemcellulare to oxidative stress

The adaptive response of the phytopathogenic fungus Fusarium decemcellulare to the oxidative stress induced by hydrogen peroxide and juglone (5-hydroxy-1,4-naphthoquinone) was studied. At concentrations higher than 1 mM, H2O2 and juglone completely inhibited the growth of the fungus. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H2O2 (0.25 mM) and juglone (0.1 mM) led to the development of a resistance to high concentrations of these oxidants. The stationary-phase cells were found to be more resistant to the oxidants than the logarithmic-phase cells. The adaptation of fungal cells to H2O2 and juglone was associated with an increase in the activity of cellular catalase and superoxide dismutase, the main oxidative stress defense of enzymes.     

Tolerance of the yeast Yarrowia lipolytica to oxidative stress

The adaptive response of the yeast Yarrowia lipolytica to the oxidative stress induced by the oxidants hydrogen peroxide, menadione, and juglone has been studied. H2O2, menadione, and juglone completely inhibited yeast growth at concentrations higher than 120, 0.5, and 0.03 mM, respectively. The stationary-phase yeast cells were found to be more resistant to the oxidants than the exponential-phase cells. The 60-min pre-treatment of logarithmic-phase cells with nonlethal concentrations of H2O2 (0.3 mM), menadione (0.05 mM), and juglone (0.005 mM) made the cells more resistant to high concentrations of these oxidants. The adaptation of yeast cells to H2O2, menadione, and juglone was associated with an increase in the activity of cellular catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase, the main enzymes involved in cell defense against oxidative stress.     

Adaptation of the Phytopathogenic Fungus Fusarium decemcellulare to Oxidative Stress

The adaptive response of the phytopathogenic fungus Fusarium decemcellulare to the oxidative stress induced by hydrogen peroxide and juglone (5-hydroxy-1,4-naphthoquinone) was studied. At concentrations higher than 1 mM, H₂O₂ and juglone completely inhibited the growth of the fungus. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H₂O₂ (0.25 mM) and juglone (0.1 mM) led to the development of a resistance to high concentrations of these oxidants. The stationary-phase cells were found to be more resistant to the oxidants than the logarithmic-phase cells. The adaptation of fungal cells to H₂O₂ and juglone was associated with an increase in the activity of cellular catalase and superoxide dismutase, the main enzymes involved in the defense against oxidative stress.     

Tolerance of the yeast Yarrowia lipolytica to oxidative stress

The adaptive response of the yeast Yarrowia lipolytica to the oxidative stress induced by the oxidants hydrogen peroxide, menadione, and juglone has been studied. H₂O₂, menadione, and juglone completely inhibited yeast growth at concentrations higher than 120, 0.5, and 0.03 mM, respectively. The stationary-phase yeast cells were found to be more resistant to the oxidants than the exponential-phase cells. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H₂O₂ (0.3 mM), menadione (0.05 mM), and juglone (0.005 mM) made the cells more resistant to high concentrations of these oxidants. The adaptation of yeast cells to H₂O₂, menadione, and juglone was associated with an increase in the activity of cellular catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase, the main enzymes involved in cell defense against oxidative stress.     

Respiratory activity of yeast Yarrowia lipolytica under oxidative stress and heat shock

Heat shock (45 degrees C) and the effect of oxidants (H2O2) resulted in a decrease of the respiratory activity of yeast cells and their survival rate. Increased resistance to stress effects after mild heat treatment (37 degrees C) or treatment with a nonlethal dose of oxidants (0.5 mM H2O2 for 60 min) was accompanied by appearance of an alternative (cyanide-resistant) oxidative pathway in the mitochondria, which promotes survival due to retention of the capacity for ATP synthesis in the first coupling point at the level of endogenous NADH dehydrogenase. The alternative oxidative pathway is more resistant to the effect of stressors that disrupt electron transfer in the cytochrome site of the respiratory chain.     

Metabolism of cyanide by Phanerochaete chrysosporium

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 (LiP H2) from the white rot fungus Phanerochaete chrysosporium was strongly inhibited by sodium cyanide. The I50 was estimated to be about 2-3 microM. In contrast, sodium cyanide binds to the native enzyme with an apparent sodium cyanide dissociation constant Kd of about 10 microM. Inhibition of the veratryl alcohol oxidase activity of LiP H2 by cyanide was reversible. Ligninolytic cultures of P. chrysosporium mineralized cyanide at a rate that was proportional to the concentration of cyanide to 2 mM. The N-tert-butyl-alpha-phenylnitrone-cyanyl radical adduct was observed by ESR spin trapping upon incubation of LiP H2 with H2O2 and sodium cyanide. The identity of the spin adduct was confirmed using 13C-labeled cyanide. Six-day-old cultures of the fungus were more tolerant to sodium cyanide toxicity than spores. Toxicity measurements were based on the effect of sodium cyanide on respiration of the fungus as determined by the metabolism of [14C]glucose to [14C]CO2. We propose that this tolerance of the mature fungus was due to its ability to mineralize cyanide and that this fungus might be effective in treating environmental pollution sites contaminated with cyanide.     

The O2-dependence of respiration and H2O2 production in the parasitic nematode Ascaridia galli.

1. Respiration in the parasitic nematode worm Ascaridia galli was inhibited at O2 concentrations in excess of 255 microM, and an apparent Km,O2 of 174 microM was determined. 2. Mitochondria-enriched fractions isolated from the tissues of A. galli have much lower apparent Km,O2 values (approx. 5 microM). They produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 3. Antimycin A inhibited respiration in muscle tissue mitochondria by 10%, but had no effect on respiration in gut + reproductive tissue mitochondria; the major portion of respiration in both types of mitochondria could be attributed to an alternative electron-transport pathway. 4. o-Hydroxydiphenyl, an inhibitor of alternative electron-transport pathways, inhibits respiration by 98% and completely inhibits the production of H2O2 in gut-plus-reproductive-tissue mitochondria; respiration and H2O2 production in muscle tissue mitochondria were inhibited by 90 and 86% respectively. 5. Another inhibitor of alternative electron transport, salicylhydroxamic acid, had the same effect as o-hydroxydiphenyl on H2O2 production and respiration in gut-plus-reproductive-tissue mitochondria. However, its effect on muscle tissue mitochondria was complex; a low concentration (0.35 mM) stimulated H2O2 production, whereas 3 mM inhibited respiration by 87% and prevented H2O2 production completely. 6. The similarities between the apparent Km,O2 values for H2O2 production and respiration in muscle mitochondria and in gut-plus-reproductive-tissue mitochondria suggests that the site of H2O2 production on the alternative electron-transport chain is cytochrome 'o'. 7. These results are discussed in relation to potential O2 toxicity in A. galli.     

Effects of hydrogen peroxide on MAPK activation, IL-8 production and cell viability in primary cultures of human bronchial epithelial cells

The airway epithelium is continuously exposed to inhaled oxidants, including airborne pollutants and cigarette smoke, which can exert harmful proinflammatory and cytotoxic effects. Therefore, the aim of our study was to investigate, in primary cultures of human bronchial epithelial cells (HBEC), the signal transduction pathways activated by increasing concentrations (0.25, 0.5, and 1 mM) of hydrogen peroxide (H(2)O(2)), as well as their effects on IL-8 production and cell viability. The reported results show that H(2)O(2) elicited, in a concentration-dependent fashion, a remarkable increase in phosphorylation-dependent activation of mitogen-activated protein kinases (MAPKs), associated with a significant induction of IL-8 synthesis and a dramatically enhanced cell death. Pre-treatment of HBEC with MAPK inhibitors was able to significantly inhibit the effects of H(2)O(2) on IL-8 secretion, and to effectively prevent cell death. Therefore, these findings suggest that MAPKs play a key role as molecular transducers of the airway epithelial injury triggered by oxidative stress, as well as potential pharmacologic targets for indirect antioxidant intervention.     

Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells.

The thioredoxin/thioredoxin reductase system has been studied as regenerative machinery for proteins inactivated by oxidative stress in vitro and in cultured endothelial cells. Mammalian glyceraldehyde-3-phosphate dehydrogenase was used as the main model enzyme for monitoring the oxidative damage and the regeneration. Thioredoxin and its reductase purified from bovine liver were used as the regenerating system. The physiological concentrations (2-14 microM) of reduced thioredoxin, with 0.125 microM thioredoxin reductase and 0.25 mM NADPH, regenerated H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase and other mammalian enzymes almost completely within 20 min at 37 degrees C. Although the treatment of endothelial cells with 0.2-12 mM H2O2 for 5 min resulted in a marked decrease in the activity of glyceraldehyde-3-phosphate dehydrogenase, it had no effect on the activities of thioredoxin and thioredoxin reductase. Essentially all of the thioredoxin in endothelial cells at control state was in the reduced form and 70-85% remained in the reduced form even after the H2O2 treatment. The inactivated glyceraldehyde-3-phosphate dehydrogenase in a cell lysate prepared from the H2O2-treated endothelial cells was regenerated by incubating the lysate with 3 mM NADPH at 37 degrees C and the antiserum raised against bovine liver thioredoxin inhibited the regeneration. The inhibition of thioredoxin reductase activity by 13-cis-retinoic acid resulted in a decrease in the regeneration of glyceraldehyde-3-phosphate dehydrogenase in the H2O2-treated endothelial cells. The present findings provide evidence that thioredoxin is involved in the regeneration of proteins inactivated by oxidative stress in endothelial cells.     

Respiratory activity of yeast Yarrowia lipolytica under oxidative stress and heat shock

Heat shock (45°C) and the effect of oxidants (H₂O₂) resulted in a decrease of the respiratory activity of yeast cells and their survival rate. Increased resistance to stress effects after mild heat treatment (37°C) or treatment with a nonlethal dose of oxidants (0.5 mM H₂O₂) for 60 min) was accompanied by appearance of an alternative (cyanide-resistant) oxidative pathway in the mitochondria, which promotes survival due to retention of the capacity for ATP synthesis in the first coupling point at the level of endogenous NADH dehydrogenase. The alternative oxidative pathway is more resistant to the effect of stressors that disrupt electron transfer in the cytochrome site of the respiratory chain.     

The role of glutathione, membrane sphingomyelin, and its metabolites in oxidative stress-induced calcium "dysregulation" in PC12 cells

Previous research showed that increasing membrane sphingomyelin (SPH) levels in rat pheochromocytoma (PC12) cells to the same extent as that seen in some brain regions with aging dramatically increases the vulnerability to oxidative stress (OS). These increases in vulnerability were determined by assessing deficits in the ability of these cells to extrude and/or sequester Ca2+ following 30 mM KCl-induced depolarization (recovery). The purpose of the present experiments was to discern whether increasing the levels of particular SPH metabolite(s), i.e., ceramide (Cer), sphingosine (Ssine), or sphingosine-1-phosphate (SPP), or indirectly increasing the concentrations of these metabolites with sphingomylinase (Sase), would interact with the cell's sensitivity to OS induced by low (5 microM) or high (nonlethal, 300 microM) H2O2. In addition, the OS vulnerability was examined as above under decreased SPH levels by exposing the cells to L-cycloserine (Lcc), which prevents SPH synthesis. Both Sase and SPP significantly decreased Ca2+ recovery of PC12 cells after H2O2 exposure. Conversely, Lcc-treated cells showed no further OS-induced decrements in recovery below those seen in controls. SPP significantly decreased glutathione levels (GSH) in the absence of OS. Repletion of GSH with 20 mM N-acetylcysteine significantly attenuated the effect of 5 microM H2O2 on recovery in SPP-treated cells and decreased sensitivity of SPP-treated cells to low doses of OS. Overall, our results suggest a critical role for GSH and SPP in the regulation of OS vulnerability, especially as it relates to Ca2+ homeostasis.     

Oxidative phosphorylation in nuclei isolated from rat thymus

1. Nuclei were isolated from rat thymus in 0.25 M sucrose-3 mM CaCl₂. Fractionation of thymus tissue showed that the nuclear fraction could be contaminated with 10% intact cells at the most.

2. The nuclear fraction contained about 50% of the cellular ATP. Under anaerobic conditions nuclear ATP is degraded. When, after an anaerobic incubation, the nuclei are incubated under aerobic conditions endogenous nuclear ATP is resynthesized. The amount of ATP found cannot be accounted for by mitochondria or intact cells. It could also be shown that the ATP synthesis is not due to such contamination.

3. Nuclear ATP synthesis and respiration were inhibited by amytal, rotenone, and carbon monoxide. The latter inhibition was reversed by light. 2,4-Dinitrophenol abolished ATP synthesis and accelerated respiration. Oligomycin inhibited respiration and phosphorylation. The inhibition of respiration by oligomycin could be released by dinitrophenol. ATP synthesis was also inhibited by lewisite. This inhibition could be reversed by BAL. No inhibition, however, was found with arsenite or arsenite + BAL.

4. A P:O ratio between 0.6 and 1.0 was obtained for nuclear phosphorylation. This must be considered as a minimal value.

5. It is concluded that thymus nuclei are capable of respiratory-chain-linked oxidative phosphorylation.     

Resistance of Penicillium piceum F-648 to hydrogen peroxide under short term and prolonged oxidative stress

Resistance of Penicillium piceum F-648 to hydrogen peroxide under short-term and prolonged oxidative stress was studied. An increase in the activity of intracellular catalase in fungal cells after short-term exposure to hydrogen peroxide was shown. Activation of fungal cells induced by H2O2 depends on H2O2 concentration, time of exposure, and the growth phase of the fungus. Variants of P. piceum F-648 that produced two forms of extracellular catalase with different catalytic properties were obtained due to prolonged adaptation to H2O2. Catalase with low affinity for substrate was produced predominantly by the parent culture and variant 3; however, a high substrate affinity of catalase was observed in variant 5. Variant 5 of P. peniceum F-648 displayed a high catalytic activity and operational stability of catalase in the presence of phosphate ions and the concentration of substrate less than 30 mM at pH more than 7.     

Hydrogen peroxide suppresses U937 cell death by two different mechanisms depending on its concentration

To investigate the mechanisms of H2O2 adaptation in mammalian cells, we exposed human U937 leukemia cells to 0.05 mM H2O2. This treatment significantly suppressed cell death and DNA fragmentation induced by a subsequent challenge with 1 mM H2O2. A more dramatic protection was observed when cells were pretreated with 0.25 mM H2O2. Pretreatment with either 0.05 or 0.25 mM H2O2 also imparted cells with a survival advantage against serum withdrawal and C2-ceramide treatment. H2O2 was found to be a mediator of cell death induced by serum withdrawal, but not by the addition of C2-ceramide. Interestingly, 0.25 mM H2O2 greatly induced glutathione peroxidase, a H2O2-consuming enzyme, whereas 0.05 mM H2O2 did not. Consistent with observation, pretreatment with 0.25 mM H2O2 resulted in a great reduction of cellular oxidant levels as determined by 2'7'-dichlorofluorescein fluorescence, and it also prevented elevation of oxidant levels upon subsequent challenge with 1 mM H2O2 or with serum withdrawal. These effects were not observed in cells pretreated with 0.05 mM H2O2. The sum of the data indicated that H2O2 suppresses cell death by two different mechanisms depending on its concentration: Relatively high concentrations enhance cellular antioxidant capacity, and lower concentrations block the lethal action of H2O2.     

Hydrogen peroxide production in uncoupled mitochondria of the parasitic nematode worm Nippostrongylus brasiliensis.

1. Mitochondria from the parasitic nematode worm Nippostrongylus brasiliensis produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 2. Antimycin A inhibits respiration and H2O2 production by 70 and 65% respectively; the residual activities can be attributed to alternative electron-transport pathway(s). 3. o-Hydroxydiphenyl and 1,3,5-trihydroxybenzene, inhibitors of alternative electron transport, inhibit respiration by 37% and H2O2 production by 26%. 4. Another inhibitor of alternative electron transport, salicylhydroxamic acid, shows a complex mode of action; low concentrations (less than 0.5 mM) stimulate respiration and H2O2 production, whereas 2 mM-salicylhydroxamic acid inhibited respiration by 35% and stopped H2O2 production completely. 5. O2 thresholds were observed for the inhibition of respiration at O2 concentrations greater than 57.7 microM and inhibition of H2O2 production (greater than 20.5 microM-O2); apparent Km values for oxygen were 5.5 microM and 3.0 microM respectively. 6. In the presence of antimycin A the O2-inhibition thresholds and apparent Km values for O2 of respiration and H2O2 production matched closely, suggesting that the alternative oxidase is a likely site of H2O2 production. 7. These results are discussed in relation to O2 toxicity to N. brasiliensis.     

Induction of a hypermetabolic state in cultured hepatocytes by glucagon and H2O2

Stress hormones and pro-inflammatory cytokines are putative signals triggering increased energy expenditure or "hypermetabolism" commonly observed in inflammatory states. Cytokines also cause the release of reactive oxidants by immune cells resident in tissues in vivo. Therefore, we hypothesized that oxidative stress plays a role in the induction of hypermetabolism. We examined the effect of glucagon (1.0 nM), a catabolic stress hormone, and the oxidant H(2)O(2) (1.0 mM) on the metabolism of stable hepatocyte cultures for 4 days. Combined H(2)O(2) and glucagon treatment, but not H(2)O(2) or glucagon used alone, increased the hepatocyte oxygen uptake rate 25% above control untreated cells after a lag-time of 72 h. The same treatment also increased the expression of mitochondrial uncoupling protein-2 (UCP2). These effects were significantly inhibited by the antioxidant N-acetylcysteine (5mM) and the pentose phosphate pathway (PPP) inhibitor dehydroepianderosterone (200 microM). Glucagon alone induced urea synthesis and H(2)O(2) alone induced the PPP. These findings show, for the first time, that oxidative stress, in combination with glucagon, increases metabolic energy expenditure in cultured cells, and that this effect may be mediated by UCP-2. Furthermore, the results implicate the PPP in the induction of the hypermetabolic response.     

Stimulated human neutrophils damage cardiac sarcoplasmic reticulum function by generation of oxidants

An important aspect of myocardial injury is the role of neutrophils in post-ischemic damage to the heart. Stimulated neutrophils initiate a series of reactions that produce toxic oxidizing agents. Superoxide rapidly dismutases to H2O2 and neutrophils contain myeloperoxidase which catalyzes the oxidation of Cl- by H2O2 to yield hypochlorous acid (HOCl). The highly reactive HOCl combines non-enzymatically with nitrogenous compounds to generate long-lived, non-radical oxidants, monochloramine and taurine N-monochloramine. We investigated the role of oxygen radicals and long-lived oxidants on cardiac sarcoplasmic reticulum function, which plays a major role in the regulation of intracellular Ca2+ and thereby in the generation of force. Incubation of sarcoplasmic reticulum with phorbol myristate acetate (PMA)-stimulated neutrophils (4 x 10(6) cells/ml) significantly decreased calcium uptake rate (0.85 +/- 0.11 to 0.11 +/- 0.06 mumol/min per mg) and Ca2+-ATPase activity (1.67 +/- 0.08 to 0.46 +/- 0.10 mumol/min per mg). Inclusion of myeloperoxidase inhibitors (cyanide, sodium azide and 3-amino-1,2,4-triazole), catalase, superoxide dismutase plus catalase, and alpha-tocopherol significantly protected (P less than 0.01) calcium uptake rates and Ca2+-ATPase activity of sarcoplasmic reticulum. Superoxide dismutase (10 microgram/ml) alone or deferoxamine (1 mM) had no protective effect in this system. The maximum inhibition of sarcoplasmic reticulum function was observed with (3-4) x 10(6) cells/ml in 4-6 min. HOCl and NH2Cl inhibited calcium uptake rate and Ca2+-ATPase activity of sarcoplasmic reticulum in a dose-dependent manner (2-20 microM), whereas H2O2 damaged sarcoplasmic reticulum at concentrations ranging from 5 to 25 mM. HOCl (20 microM) inhibited 80-90% of Ca2+-uptake rate and Ca2+-ATPase activity and L-methionine (0.1-1 mM) provided complete protection. We conclude that stimulated neutrophils damage cardiac sarcoplasmic function by generation of myeloperoxidase-catalyzed oxidants.     

Studies on the mechanism of NADPH oxidation by the granule fraction isolated from human resting polymorphonuclear blood cells.

Various factor affecting NADPH-oxidation by resting human leucocyte granules (LG) at acid pH, have been investigated. It was found that: 1) oxidation of NADPH by LG was increasingly inhibited by increased cyanide concentrations in the medium and was abolished by 4 mM cyanide. 2) with or without cyanide in the incubation medium, LG omitted, Mn++ in the presence of NADPH induced superoxide anion (O- WITH 2) production, as evidenced by oxygen consumption and H2O2 production, which were abolished (in the absence of cyanide) by cytochrome C (a potent O- with 2 scavenger). 3) Both NADPH oxidation in the presence of 2 mM cyanide (cyanide-resistant) and in its absence (cyanide-sensitive) by LG occurred only in the presence of Mn++, and both were inhibited by superoxide dismutase. 4) Cyanide-resistant NADPH oxidation by LG generated H2O2, was inhibited by H2O2 and was not modified by "active" catalase. The ratio of cyanide-resistant NADPH oxidation/O2 uptake was 1 up to 1.25 mM NADPH, and increased above this concentration. 5) Cyanide-sensitive NADPH oxidation was inhibited by catalase and increased upon addition of H2O2. The ratio of cyanide-sensitive NADPH oxidation/O2 uptake was 2. It was concluded that after initiation by O - with 2, produced independently of LG, two sequential types of LG dependent NADPH oxidations occur. First, an O - with 2-dependent protein mediated NADPH oxidation (cyanide-resistant) which generates H2O2 and O - with 2 occurs. Second, NADPH peroxidation (cyanide-sensitive) which utilizes H2O2 takes place.     

Respiration in the filarial nematode Brugia pahangi

Glucose-supported O2 uptake in the filarial nematode Brugia pahangi was partially inhibited by antimycin A (30-40%), with the remaining activity being sensitive to o-hydroxydiphenyl or salicylhydroxamic acid (SHAM). The production of CO2 by B. pahangi in the presence of D-glucose was stimulated by O2; the stimulation of CO2; the stimulation of CO2 production was sensitive to antimycin A. The O2 dependencies of respiration showed that the apparent O2 affinity for B. pahangi was diminished in the presence of antimycin A; O2 thresholds for inhibition of respiration were observed which showed that the alternative electron transport pathway was less sensitive to inhibition at elevated O2 concentrations. H2O2 production and its excretion could be detected in whole B. pahangi; higher rates were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. The effects of inhibitors on H2O2 production suggest two sites of H2O2 production, one associated with the classical antimycin A-sensitive pathway, the other with the alternative respiratory pathway. The similarity in the O2 dependencies of H2O2 production and respiration may indicate that H2O2 production is involved in O2-mediated toxicity. Succinate and malate respiring sub-mitochondrial particles of B. pahangi produced O2.- radicals at a site on the antimycin A-sensitive respiratory pathway. Inhibition of the alternative electron pathway by SHAM was unusual; sub-millimolar concentrations markedly stimulated respiration, H2O2 production and O2.- production by 30, 20 and 25%, respectively, whereas higher concentrations (greater than 2.5 mM) inhibited respiration by 75% and H2O2 and O2.- production by up to 85%.