Hypoxia tolerance in animals: biology and application.

Many invertebrates and ectothermic vertebrates successfully cope with a fluctuating supply of ambient oxygen-and consequently, a highly variable tissue oxygenation-through increasing their antioxidant barriers. During chronic deprivation of oxygen, however, the hypometabolic defense mode of the fruit fly Drosophila, the hypoxia-induced behavioral hypothermia of the crayfish Pacifastacus leniusculus, and the production of ethanol during anoxia by the crucian carp Carassius carassius all indicate that these animals are also capable of utilizing a suite of genetic and physiological defenses to survive otherwise lethal reductions in tissue oxygenation. Normally, much of an organism's gene response to hypoxia is orchestrated via the hypoxia-inducible transcription factor HIF. Recent developments expand our view of HIF function even further by highlighting regulatory roles for HIF in the hypometabolism of insects, in the molting and the normoxic immune response of crustaceans, and in the control-via the downstream effector gene erythropoietin-of the hypoxic ventilatory response and pulmonary hypertension in mammals. These and related topics were collectively presented by the authors in a symposium of the 2008 ICA-CBP conference at Mara National Reserve, Kenya, Africa. This synthesis article communicates the essence of the symposium presentations to the wider community.

The effect of buthionine sulfoximine on the glutathione level in goldfish tissues.

This study describes the effect of DL-buthionine-[S,R]-sulfoximine (BSO) on the glutathione equivalents (GSH-eq = GSH + 2 GSSG) of goldfish. BSO causes depletion of cellular GSH by inhibiting gamma-glutamylcysteine synthetase, a key enzyme of the GSH biosynthesis pathway. BSO at 1,000 and 1,500 mg/kg was effective in promoting 50 and 80% depletion of GSH-eq from brain and liver, respectively, within 3 days. Lower doses of BSO failed to effectively promote hepatic GSH-eq depletion. Moreover, no evident toxic side-effects were observed (including hepatic lipid peroxidation and free radical-mediated oxidation of proteins) in goldfish in response to BSO intraperitoneal injections. We conclude that BSO can be used to deplete GSH-eq in goldfish liver and brain, but attention should be paid to species-specific variations in BSO effects.

Oxidative stress and antioxidant defenses in goldfish Carassius auratus during anoxia and reoxygenation.

The purpose of this work was to evaluate the response of the antioxidant system of goldfish Carassius auratus during anoxia and reoxygenation. The exposure of goldfish to 8 h of anoxia induced a 14% decrease in total glutathione levels in the kidney, although the liver, brain, and muscle were unaffected. Anoxia also resulted in increases in the activities of liver catalase, brain glucose-6-phosphate dehydrogenase, and brain glutathione peroxidase (by 38, 26, and 79%, respectively) and a decrease in kidney catalase activity (by 17.5%). After 14 h of reoxygenation, liver catalase and brain glutathione peroxidase activities remained higher than controls and several other tissue-specific changes occurred in enzyme activities. Superoxide dismutase activity was unaffected by anoxia and reoxygenation. The levels of conjugated dienes, as indicators of lipid peroxidation, increased by 114% in liver after 1 h of reoxygenation and by 75% in brain after 14 h of reoxygenation. Lipid peroxidation was unaffected in kidney and depressed during anoxia and reoxygenation (by 44-61%) in muscle. Regulation of the goldfish antioxidant system during anoxia may constitute a biochemical mechanism that minimizes oxidative stress following reoxygenation.

The iron chelator pyridoxal isonicotinoyl hydrazone inhibits mitochondrial lipid peroxidation induced by Fe(II)-citrate.

Pyridoxal isonicotinoyl hydrazone (PIH) is able to prevent iron-mediated hydroxyl radical formation by means of iron chelation and inhibition of redox cycling of the metal. In this study, we investigated the effect of PIH on Fe(II)-citrate-mediated lipid peroxidation and damage to isolated rat liver mitochondria. Lipid peroxidation was quantified by the production of thiobarbituric acid-reactive substances (TBARS) and by antimycin A-insensitive oxygen consumption. PIH at 300 microM induced full protection against 50 microM Fe(II)-citrate-induced loss of mitochondrial transmembrane potential (deltapsi) and mitochondrial swelling. In addition, PIH prevented the Fe(II)-citrate-dependent formation of TBARS and antimycin A-insensitive oxygen consumption. The antioxidant effectiveness of 100 microM PIH (on TBARS formation and mitochondrial swelling) was greater in the presence of 20 or 50 microM Fe(II)-citrate than in the presence of 100 microM Fe(II)-citrate, suggesting that the mechanism of PIH antioxidant action is linked with its Fe(II) chelating property. Finally, PIH increased the rate of Fe(II) autoxidation by sequestering iron from the Fe(II)-citrate complex, forming a Fe(III)-PIH, complex that does not participate in Fenton-type reactions and lipid peroxidation. These results are of pharmacological relevance since PIH is a potential candidate for chelation therapy in diseases related to abnormal intracellular iron distribution and/or iron overload.

Pyridoxal isonicotinoyl hydrazone (PIH) prevents copper-mediated in vitro free radical formation.

Pyridoxal isonicotinoyl hydrazone (PIH) is an iron chelator with antioxidant activity, low toxicity and is useful in the experimental treatment of iron-overload diseases. Previous studies on x-ray diffraction have revealed that PIH also forms a complex with Cu(II). Since the main drug of choice for the treatment of Wilson's disease, d-penicillamine, causes a series of side effects, there is an urgent need for the development of alternative copper chelating agents for clinical use. These chelators must also have antioxidant activity because oxidative stress is associated with brain and liver copper-overload. In this work we tested the ability of PIH to prevent in vitro free radical formation mediated by Cu(II), ascorbate and dissolved O2. Degradation of 2-deoxyribose mediated by 10 microM Cu(II) and 3 mM ascorbate was fully inhibited by 10 microM PIH (I50 = 6 microM) or 20 microM d-penicillamine (I50 = 10 microM). The antioxidant efficiency of PIH remained unchanged with increasing concentrations (from 1 to 15 mM) of the hydroxyl radical detector molecule, 2-deoxyribose, indicating that PIH does not act as a hydroxyl scavenger. On the other hand, the efficiency of PIH (against copper-mediated 2-deoxyribose degradation and ascorbate oxidation) was inversely proportional to the Cu(II) concentration, suggesting a competition between PIH and ascorbate for complexation with Cu(lI). An almost full inhibitory effect by PIH was observed when the ratio PIH:copper was 1:1. A similar result was obtained with the measurement of copper plus ascorbate-mediated O2 uptake. Moreover, spectral studies of the copper and PIH interaction showed a peak at 455 nm and also indicated the formation of a stable Cu(II) complex with PIH with a 1:1 ratio. These data demonstrated that PIH prevents hydroxyl radical formation and oxidative damage to 2-deoxyribose by forming a complex with Cu(II) that is not reactive with ascorbate (first step of the reactions leading to hydroxyl radical formation from Cu(II), ascorbate and O2) and does not participate in Haber-Weiss reactions.

The iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and its analogues prevent damage to 2-deoxyribose mediated by ferric iron plus ascorbate.

Iron chelating agents are essential for treating iron overload in diseases such as beta-thalassemia and are potentially useful for therapy in non-iron overload conditions, including free radical mediated tissue injury. Deferoxamine (DFO), the only drug available for iron chelation therapy, has a number of disadvantages (e.g., lack of intestinal absorption and high cost). The tridentate chelator pyridoxal isonicotinoyl hydrazone (PIH) has high iron chelation efficacy in vitro and in vivo with high selectivity and affinity for iron. It is relatively non-toxic, economical to synthesize and orally effective. We previously demonstrated that submillimolar levels of PIH and some of its analogues inhibit lipid peroxidation, ascorbate oxidation, 2-deoxyribose degradation, plasmid DNA strand breaks and 5,5-dimethylpyrroline-N-oxide (DMPO) hydroxylation mediated by either Fe(II) plus H(2)O(2) or Fe(III)-EDTA plus ascorbate. To further characterize the mechanism of PIH action, we studied the effects of PIH and some of its analogues on the degradation of 2-deoxyribose induced by Fe(III)-EDTA plus ascorbate. Compared with hydroxyl radical scavengers (DMSO, salicylate and mannitol), PIH was about two orders of magnitude more active in protecting 2-deoxyribose from degradation, which was comparable with some of its analogues and DFO. Competition experiments using two different concentrations of 2-deoxyribose (15 vs. 1.5 mM) revealed that hydroxyl radical scavengers (at 20 or 60 mM) were significantly less effective in preventing degradation of 2-deoxyribose at 15 mM than 2-deoxyribose at 1.5 mM. In contrast, 400 microM PIH was equally effective in preventing degradation of both 15 mM and 1.5 mM 2-deoxyribose. At a fixed Fe(III) concentration, increasing the concentration of ligands (either EDTA or NTA) caused a significant reduction in the protective effect of PIH towards 2-deoxyribose degradation. We also observed that PIH and DFO prevent 2-deoxyribose degradation induced by hypoxanthine, xanthine oxidase and Fe(III)-EDTA. The efficacy of PIH or DFO was inversely related to the EDTA concentration. Taken together, these results indicate that PIH (and its analogues) works by a mechanism different than the hydroxyl radical scavengers. It is likely that PIH removes Fe(III) from the chelates (either Fe(III)-EDTA or Fe(III)-NTA) and forms a Fe(III)-PIH(2) complex that does not catalyze oxyradical formation.

Polyphenol tannic acid inhibits hydroxyl radical formation from Fenton reaction by complexing ferrous ions.

Tannic acid (TA), a plant polyphenol, has been described as having antimutagenic, anticarcinogenic and antioxidant activities. Since it is a potent chelator of iron ions, we decided to examine if the antioxidant activity of TA is related to its ability to chelate iron ions. The degradation of 2-deoxyribose induced by 6 microM Fe(II) plus 100 microM H2O2 was inhibited by TA, with an I50 value of 13 microM. Tannic acid was over three orders of magnitude more efficient in protecting against 2-deoxyribose degradation than classical *OH scavengers. The antioxidant potency of TA was inversely proportional to Fe(II) concentration, demonstrating a competition between H2O2 and AT for reaction with Fe(II). On the other hand, the efficiency of TA was nearly unchanged with increasing concentrations of the *OH detector molecule, 2-deoxyribose. These results indicate that the antioxidant activity of TA is mainly due to iron chelation rather than *OH scavenging. TA also inhibited 2-deoxyribose degradation mediated by Fe(III)-EDTA (iron = 50 microM) plus ascorbate. The protective action of TA was significantly higher with 50 microM EDTA than with 500 microM EDTA, suggesting that TA removes Fe(III) from EDTA and forms a complex with iron that cannot induce *OH formation. We also provided evidence that TA forms a stable complex with Fe(II), since excess ferrozine (14 mM) recovered 95-96% of the Fe(II) from 10 microM TA even after a 30-min exposure to 100-500 microM H2O2. Addition of Fe(III) to samples containing TA caused the formation of Fe(II)n-TA, complexes, as determined by ferrozine assays, indicating that TA is also capable of reducing Fe(III) ions. We propose that when Fe(II) is complexed to TA, it is unable to participate in Fenton reactions and mediate *OH formation. The antimutagenic and anticarcinogenic activity of TA, described elsewhere, may be explained (at least in part) by its capacity to prevent Fenton reactions.

Role of Fe(III) in Fe(II)citrate-mediated peroxidation of mitochondrial membrane lipids.

In this report we study the effect of Fe(III) on lipid peroxidation induced by Fe(II)citrate in mitochondrial membranes, as assessed by the production of thiobarbituric acid-reactive substances and antimycin A-insensitive oxygen uptake. The presence of Fe(III) stimulates initiation of lipid peroxidation when low citrate:Fe(II) ratios are used (< or = 4:1). For a citrate:total iron ratio of 1:1 the maximal stimulation of lipid peroxidation by Fe(III) was observed when the Fe(II):Fe(III) ratio was in the range of 1:1 to 1:2. The lag phase that accompanies oxygen uptake was greatly diminished by increasing concentrations of Fe(III) when the citrate:total iron ratio was 1:1, but not when this ratio was higher. It is concluded that the increase of lipid peroxidation by Fe(III) is observed only when low citrate:Fe(II) ratios were used. Similar results were obtained using ATP as a ligand of iron. Monitoring the rate of spontaneous Fe(II) oxidation by measuring oxygen uptake in buffered medium, in the absence of mitochondria, Fe(III)-stimulated oxygen consumption was observed only when a low citrate:Fe(II) ratio was used. This result suggests that Fe(III) may facilitate the initiation and/or propagation of lipid peroxidation by increasing the rate of Fe(II)citrate-generated reactive oxygen species.

Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro.

Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor. This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciola hepatica and Schistosoma mansoni. Using partially purified cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro. A system containing FeIII-EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxyl radical. Fasciola hepatica cytochrome c peroxidase effectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c. This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c. Due to the low specific activity of the final partially purified sample the effects of ascorbate and calcium chloride on cytochrome c peroxidase were investigated. The activity of the partially purified enzyme was found to increase between 10 and 37% upon reduction with ascorbate. However, incubation of the partially purified enzyme with 1 mM calcium chloride did not have any effect on enzyme activity. Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of the highly toxic hydroxyl radical (.OH). We suggest that the capacity of CcP to inhibit .OH-formation, by efficiently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica.

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on *OH formation by the Fenton reaction.

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.

The iron chelator pyridoxal isonicotinoyl hydrazone (PIH) protects plasmid pUC-18 DNA against *OH-mediated strand breaks.

Pyridoxal isonicotinoyl hydrazone (PIH) has previously been studied for use in iron chelation therapy in iron-overload diseases. It is an efficient in vitro antioxidant due to its Fe(III) complexing activity (Schulman, H. M., et al. Redox Report 1:373-378; 1995). Pathologies associated with iron-overload include hepatic and other cancers. Since oxidative alterations of DNA can be linked to the development of cancer, we decided to study whether PIH protects DNA against in vitro oxidative stress. We report here that pUC-18 plasmid DNA is damaged by *OH radicals generated from Fe(II) plus H2O2 or from Fe(II) plus hypoxanthine/xanthine oxidase. The DNA damage was quantified by determining the diminution of supercoiled DNA forms after oxidative attack using agar gel electrophoresis. Micromolar amounts of PIH (20-30 microM) were able to half-protect DNA from iron (1-7.5 microM)-mediated *OH formation. The antioxidant capacity of PIH was significantly higher than that of some of its analogs and desferrioxamine. PIH and some of its analogues could also inhibit the oxidative degradation of 2-deoxyribose caused by Fenton reagents. Since we observed that PIH enhances the Fe(II) autoxidation rate, measured by the ferrozine technique, PIH may limit *OH formation and consequently DNA damage by decreasing the amount of Fe(II) available to catalyze Fenton reactions.

Antioxidant defenses and metabolic depression. The hypothesis of preparation for oxidative stress in land snails.

The roles of enzymatic antioxidant defenses in the natural tolerance of environmental stresses that impose changes in oxygen availability and oxygen consumption on animals is discussed with a particular focus on the biochemistry of estivation and metabolic depression in pulmonate land snails. Despite reduced oxygen consumption and PO2 during estivation, which should also mean reduced production of oxyradicals, the activities of antioxidant enzymes, such as superoxide dismutase and catalase, increased in 30 day-estivating snails. This appears to be an adaptation that allows the snails to deal with oxidative stress that takes place during arousal when PO2 and oxygen consumption rise rapidly. Indeed, oxidative stress was indicated by increased levels of lipid peroxidation damage products accumulating in hepatopancreas within minutes after arousal was initiated. The various metabolic sites responsible for free radical generation during arousal are still unknown but it seems unlikely that the enzyme xanthine oxidase plays any substantial role in this despite being implicated in oxidative stress in mammalian models of ischemia/reperfusion. We propose that the activation of antioxidant defenses in the organs of Otala lactea during estivation is a preparative mechanism against oxidative stress during arousal. Increased activities of antioxidant enzymes have also observed under other stress situations in which the actual production of oxyradicals should decrease. For example, antioxidant defenses are enhanced during anoxia exposure in garter snakes Thamnophis sirtalis parietalis (10 h at 5 degrees C) and leopard frogs Rana pipiens (30 h at 5 degrees C) and during freezing exposure (an ischemic condition due to plasma freezing) in T. sirtalis parietalis and wood frogs Rana sylvatica. It seems that enhancement of antioxidant enzymes during either anoxia or freezing is used as a preparatory mechanism to deal with a physiological oxidative stress that occurs rapidly within the early minutes of recovery during reoxygenation or thawing. Thus, a wide range of stress tolerant animals display coordinated changes in antioxidant defenses that allow them to deal with oxidative stress that occurs as part of natural cycles of stress/recovery that alter oxygen levels in tissues. The molecular mechanisms that trigger and regulate changes in antioxidant enzyme activities in these species are still unknown but could prove to have key relevance for the development of new intervention strategies in the treatment of cardiovascular ischemia/reperfusion injuries in humans.

Role of antioxidant defenses in the tolerance of severe dehydration by anurans. The case of the leopard frog Rana pipiens.

Many anurans have excellent dehydration tolerance that allows endurance of the loss of up to 50-60% of total body water. One of the effects of severe dehydration is circulatory impairment due the reduced volume and increased viscosity of blood, which leads to organ hypoxia. The rehydration situation, therefore, involves a reoxygenation of tissues that may include elements of oxidative stress that resemble the injury in post-ischemic reperfusion of mammalian organs. The role of endogenous defenses against oxygen radicals in the tolerance of severe dehydration by leopard frogs, Rana pipiens, was investigated by monitoring the activities of antioxidant enzymes and glutathione levels (reduced GSH and oxidized GSSG) in leg muscle and liver of control, 50%-dehydrated, and fully rehydrated frogs. The maximal activities of muscle catalase and liver glutathione peroxidase, measured per mg soluble protein, increased significantly by 52 and 74%, respectively, after dehydration whereas muscle superoxide dismutase and glutathione reductase activities responded oppositely, decreasing by 32 and 35%, respectively. Enzyme activities returned to control levels after full rehydration. Hepatic GSH and GSSG increased early in the rehydration process (30% recovery of total body water), but returned to control levels after full recovery. A similar trend was observed for liver GSSG. The elevation of antioxidant defenses against peroxides during dehydration could provide protection against post-hypoxic oxyradical stress during rehydration. Indeed, analysis of one product of lipid peroxidation, thiobarbituric acid reactive substances, in frog tissues gave no indication of oxidative stress during the dehydration/rehydration cycle.

Pyrophosphate and adenosine 5'-diphosphate synthesis from phospho(enol)pyruvate: catalysis by phosphate minerals and modulation by dimethyl sulfoxide.

Phospho(enol)pyruvate (PEP) undergoes transphosphorylation to form pyrophosphate (PPi) and adenosine 5'-diphosphate (5'-ADP) with high yields in the presence of an adsorbent surface of calcium phosphate (Pi.Ca), which is considered to be an ancient mineral with catalytic properties. PPi formation is a result of the phosphorolytic cleavage of the enol phosphate group of PEP by precipitated Pi. The synthesis of PPi is dependent on the amount of the solid matrix; it increases with the amount of adsorbed PEP and upon addition of dimethyl sulfoxide (Me2SO), a molecule with high dipolar moment. Although it is saturated with PEP at neutral pH, the phosphorylating Pi.Ca surface becomes effective only in alkaline conditions. In a parallel reaction, PEP phosphorylates 5'-AMP to 5'-ADP with a yield that is sevenfold higher in the presence of the Pi.Ca surface than in its absence, indicating that the solid matrix promotes interaction between adsorbed molecules with a high potential for phosphoryl transfer. In contrast to phosphorolysis, this latter reaction is stimulated by Me2SO only in homogeneous solution. It is concluded that phosphate minerals may have coadjuvated in reactions involving different phosphorylated compounds and that molecules with high dipolar moment may have acted in mildly alkaline, primitive aqueous environments to modulate phosphoryl transfer reactions catalyzed by phosphate minerals.

Relationship between anoxia exposure and antioxidant status in the frog Rana pipiens.

The biochemical adaptations of cellular antioxidant defenses that permit anoxia-tolerant animals to deal effectively with rapid and large changes in oxygen availability, and hence oxidative stress, during transitions from anoxia to normoxia provide insights into the strategies of antioxidant defense that could help to minimize reperfusion injuries to mammalian organs after anoxia/ischemia stress. The present study analyzes the effects of 30 h anoxia exposure followed by reoxygenation on the antioxidant defenses (activities of five enzymes, glutathione status) and lipid peroxidation damage to organs of the leopard frog Rana pipiens (5 degrees C-adapted autumn frogs). Exposure to 30 h anoxia resulted in significant increases in the activities of skeletal muscle and heart catalase (by 53 and 47%), heart and brain glutathione peroxidase (by 75 and 30%), and brain glutathione S-transferase (by 66%). In most cases, enzyme activities had returned to the control values after 40 h aerobic recovery. Activities of superoxide dismutase and glutathione reductase were unaltered in all of the organs, and anoxia/recovery had no effect on any of the enzymes in liver. Glutathione equivalents (GSH-eq) were maintained in four organs during anoxia but decreased by 32% in brain during anoxia. Brain GSH-eq had recovered after 90 min reoxygenation, and, in addition, hepatic GSH-eq rose by 71% after 90 min reoxygenation. The ratio of oxidized glutathione to GSH-eq was also affected by anoxia in an organ-specific way. Lipid peroxidation, assessed as the content of thiobarbituric acid-reactive substances (TBARS), was unaltered in skeletal muscle and liver after 30 h anoxia exposure or short (25 and 90 min)- or long-term (40 h) periods of reoxygenation, indicating that cycles of natural and survivable anoxia/reoxygenation occur without significant increase in TBARS in selected organs. Overall, the data demonstrate that elements of the antioxidant system of R. pipiens are induced during anoxia exposures as a possible preparation for dealing with potentially harmful oxygen reperfusion stress.

Can trifluoperazine protect mitochondria against reactive oxygen species-induced damage?

Trifluoperazine (TFP) (35 microM) prevents mitochondrial transmembrane potential (delta psi) collapse and swelling induced by 10 microM Ca2+ plus oxyradicals generated from delta-aminolevulinic acid autoxidation. In contrast with EGTA, TFP cannot restore the totally collapsed delta psi. So, TFP might not remove Ca2+ from its 'harmful site', but could impair the ROS-driven cross-linking between membrane-SH proteins. Our data are correlated with the protective uses of TFP against oxidative processes promoted by oxyradicals plus Ca2+.

Quantification of lipid peroxidation in tissue extracts based on Fe(III)xylenol orange complex formation.

Commonly used spectrophotometric methods for determining the extent of lipid peroxidation in animal tissue extracts, such as measurements of diene conjugation and thiobarbituric acid reactive substances (TBARS), have been criticized for their lack of specificity. This study shows that lipid hydroperoxides can be effectively quantified in animal tissue extracts using an assay based on the formation of a Fe(III)xylenol orange complex. Addition of H2O2, cumene hydroperoxides, or methanolic tissue extracts to an acidic reaction mixture containing 0.25 mM Fe(II) and 0.1 mM xylenol orange caused the formation of a broad Fe(III)xylenol orange complex absorbance peak at 560-580 nm with a corresponding decrease in the xylenol orange peak at 440 nm. Complex formation measured at 580 nm was saturable with both xylenol orange and Fe (II) concentration. Addition of ascorbic acid, GSH, and cysteine (0.3-5 mM) caused a saturable reduction of the Fe(III)xylenol orange complex. Formation of the Fe(III)xylenol orange complex was linear with the amount of tissue extract added. A significant correlation (r = 0.88, p < 0.005) existed between the xylenol orange method of estimating lipid peroxidation and the conventional TBARS assay in a series of animal tissues tested. The time course of increase in A580nm in tests using tissue extracts was typical of a free radical reaction; a lag phase was followed by a log phase. No increase in A580nm was observed up to 24 h when highly peroxidizable arachidonic acid was assayed. These results indicate that the formation of the Fe(III)xylenol orange complex reflects a chemical amplification of the original level of lipid hydroperoxides present in tissue extracts and that peroxidizable lipids do not influence the assay. The potential usefulness of the xylenol orange assay for comparative biochemical and toxicological studies of oxidative stress is discussed.

How do Ca2+ and 5-aminolevulinic acid-derived oxyradicals promote injury to isolated mitochondria?

The biosynthetic heme precursor 5-aminolevulinic acid (5-ALA) is a generator of oxygen radicals in vitro and possibly in vivo during pathologic situations of 5-ALA overload, for example, acute intermittent porphyria and saturnism. It has been observed that 5-ALA induces, in isolated rat liver mitochondria, permeabilization of the inner mitochondrial membrane (a phenomenon called permeability transition) as verified by the elimination of the transmembrane electrical potential, Ca2+ release, mitochondrial swelling, and increase in state-4 respiratory rate. The damaging process is primarily attributed to .OH radicals as elucidated by the protection by catalase, superoxide dismutase, and the Fe(II) chelator o-phenanthroline. Ruthenium red, EGTA, and dithiothretol (DTT) have been observed to prevent the action of 5-ALA-generated oxyradicals, suggesting the participation of both Ca2+ and the oxidation of critical thiol membrane proteins in the process of permeability transition. 5-ALA-induced polymerization of thiol membrane proteins has also been demonstrated by SDS-PAGE electrophoresis of the mitochondrial suspensions, a process similar to that observed in mitochondria treated with tert-butyI hydroperoxide. EGTA addition, in contrast with DTT or antioxidants, restores the previously eliminated electrical potential. Furthermore, EGTA prevents the 5-ALA-mediated polimeryzation of thiol proteins. These observations suggest that Ca2+ participates in a later stage of the permeability transition, after the oxidation of the thiol proteins. The effects of 5-ALA-derived oxyradicals in isolated mitochondria could be used as a tool for more general studies of oxidative stress, such as the mitochondrial injury that follows processes of ischemia and reperfusion or xenobiotic poisoning.

Xanthine oxidase and xanthine dehydrogenase from an estivating land snail.

During arousal from estivation in land snails, Otala lactea, active metabolic functions are restored within minutes and oxygen consumption increases dramatically. During the transition from the hypoxic conditions of estivation to normoxia it is possible that xanthine oxidase (XO) in hepatopancreas contributes to the observed lipid peroxidation. Using a fluorometric assay that is based on the oxidation of pterin, the activities and some properties of XO and XO+XDH (sum of XO and xanthine dehydrogenase activities) were measured in hepatopancreas extracts. Km values for pterin for XO and XO+XDH were 9 and 6 microM, respectively, and the Km of XDH for methylene blue was 5 microM. Both XO+XDH and XO activities were inhibited by allopurinol (I50 = 2 microM), pre-incubation at 40 degrees C, and by 5 min H2O2 pre-exposure. Inclusion of azide in the reaction promoted a rise of approximately 70-fold in the inactivation power of H2O2 due to inhibition of high endogenous catalase activity. The I50 for H2O2 of XO+XDH and XO activities in the presence of azide was 0.04 and 0.11 mM, respectively. Unlike the situation for mammalian XO, a previous reduction of O. lactea XO (by pterin) was not necessary to make the enzyme susceptible to H2O2 effects. Interestingly, methylene blue partially prevented both heat- and H2O2-induced inactivation of XO+XDH activity. These data indicate that the formation of an enzyme-methylene blue complex induces protection against heat and oxidative damage at the FAD-active site. Both XO and XO+XDH activites were significantly higher in snails after 35 days of estivation compared with active snails 24 h after arousal from dormancy. The ratio of XO/(XO+XDH) activities was also slightly increased in estivating O. lactea (from 0.07 to 0.09; P < 0.025). XO activity was 0.03 nmol.min-1.mg protein-1 in estivating snails. Compared with hepatopancreas catalase, XO activity is probably too low to contribute significantly to the net generation of oxyradicals, and hence to peroxidative damage. Rather, the low potential of XO to induce oxidative stress may constitute an adaptive advantage for O. lactea during arousal periods.

Antioxidant defenses and metabolic depression in a pulmonate land snail.

During arousal from estivation oxygen consumption by land snails (Otala lactea) increases severalfold. To determine whether snails prepared for an accompanying rise in the rates of oxyradical generation by altering their antioxidant defense mechanisms, changes in the activities of antioxidant enzymes and lipid peroxidation products were quantified in foot and hepatopancreas of control, 30-day estivating, and aroused snails. Compared with controls, estivating O. lactea showed significant increases in the activities of foot muscle superoxide dismutase (SOD) (increasing by 56-67%), catalase (51-72%), and glutathione S-transferase (79-108%), whereas, in hepatopancreas, SOD (57-78%) and glutathione peroxidase (93-144%) increased. Within 40 min after arousal began, hepatopancreas glutathione peroxidase activity had returned to control values, but SOD showed a further 70% increase in activity but then returned to control levels by 80 min. Estivation had no effect on total glutathione (GSH + 2 GSSG) concentrations in tissues, but GSSG content had increased about twofold in both organs of 30-day dormant snails. Lipid peoxidation (quantified as thiobarbituric acid reactive substances) was significantly enhanced at the onset of arousal from dormancy, indicating that oxidative stress and tissue damage occurred at this time. The data suggest that antioxidant defenses in snail organs are increased while snails are in the hypometabolic state as a preparation for oxidative stress during arousal.