Effect of succinate on mitochondrial lipid peroxidation. 1. Comparative studies on ferrous ion and ADP · Fe/NADPH-induced peroxidation

Lipid peroxidation in isolated rat liver mitochondria, mitoplast, and mitochondrial inner membrane fragments was induced either by ferrous ions, or in an NADPH-dependent process by complexing with adenine nucleotides (ADP or ATP) iron. The Fe²⁺-induced lipid peroxidation is nonenzymic when inner membrane fragments are used, while the differences in the inhibitory effect of Mn²⁺ ions and the stimulatory effect of the ionophore A-23187 in mitochondria and inner membrane fragments suggest an enzymic mechanism for ferrous ion-induced lipid peroxidation in intact mitochondria. Contrary to this the ADP/Fe/NADPH-dependent lipid peroxidation is an enzymic process both in mitochondria and inner membrane preparations. We have shown that cytochrome P₄₅₀ is involved in the ADP/Fe/NADPH-induced lipid peroxidation. Succinate, a known inhibitor of NADPH-dependent lipid peroxidation, inhibited the Fe²⁺-induced process also, and there was no difference in this effect when inner membrane preparations, mitochondria, or mitoplasts were used.     

An investigation into the role of hydroxyl radical in xanthine oxidase-dependent lipid peroxidation

A model lipid peroxidation system dependent upon the hydroxyl radical, generated by Fenton's reagent, was compared to another model system dependent upon the enzymatic generation of superoxide by xanthine oxidase. Peroxidation was studied in detergent-dispersed linoleic acid and in phospholipid liposomes. Hydroxyl radical generation by Fenton's reagent (FeCl₂ + H₂O₂) in the presence of phospholipid liposomes resulted in lipid peroxidation as evidenced by malondialdehyde and lipid hydroperoxide formation. Catalase, mannitol, and Tris-Cl were capable of inhibiting activity. The addition of EDTA resulted in complete inhibition of activity when the concentration of EDTA exceeded the concentration of Fe²⁺. The addition of ADP resulted in slight inhibition of activity, however, the activity was less sensitive to inhibition by mannitol. At an ADP to Fe²⁺ molar ratio of 10 to 1, 10 mm mannitol caused 25% inhibition of activity. Lipid peroxidation dependent on the enzymatic generation of superoxide by xanthine oxidase was studied in liposomes and in detergent-dispersed linoleate. No activity was observed in the absence of added iron. Activity and the apparent mechanism of initiation was dependent upon iron chelation. The addition of EDTA-chelated iron to the detergent-dispersed linoleate system resulted in lipid peroxidation as evidenced by diene conjugation. This activity was inhibited by catalase and hydroxyl radical trapping agents. In contrast, no activity was observed with phospholipid liposomes when iron was chelated with EDTA. The peroxidation of liposomes required ADP-chelated iron and activity was stimulated upon the addition of EDTA-chelated iron. The peroxidation of detergent-dispersed linoleate was also enhanced by ADP-chelated iron. Again, this peroxidation in the presence of ADP-chelated iron was not sensitive to catalase or hydroxyl radical trapping agents. It is proposed that initiation of superoxide-dependent lipid peroxidation in the presence of EDTA-chelated iron occurs via the hydroxyl radical. However, in the presence of ADP-chelated iron, the participation of the free hydroxyl radical is minimal.     

Effect of pH and Oxalate on Hydroquinone-Derived Hydroxyl Radical Formation during Brown Rot Wood Degradation

The redox cycle of 2,5-dimethoxybenzoquinone (2,5-DMBQ) is proposed as a source of reducing equivalent for the regeneration of Fe²⁺ and H₂O₂ in brown rot fungal decay of wood. Oxalate has also been proposed to be the physiological iron reductant. We characterized the effect of pH and oxalate on the 2,5-DMBQ-driven Fenton chemistry and on Fe³⁺ reduction and oxidation. Hydroxyl radical formation was assessed by lipid peroxidation. We found that hydroquinone (2,5-DMHQ) is very stable in the absence of iron at pH 2 to 4, the pH of degraded wood. 2,5-DMHQ readily reduces Fe³⁺ at a rate constant of 4.5 × 10³ M⁻¹s⁻¹ at pH 4.0. Fe²⁺ is also very stable at a low pH. H₂O₂ generation results from the autoxidation of the semiquinone radical and was observed only when 2,5-DMHQ was incubated with Fe³⁺. Consistent with this conclusion, lipid peroxidation occurred only in incubation mixtures containing both 2,5-DMHQ and Fe³⁺. Catalase and hydroxyl radical scavengers were effective inhibitors of lipid peroxidation, whereas superoxide dismutase caused no inhibition. At a low concentration of oxalate (50 μM), ferric ion reduction and lipid peroxidation are enhanced. Thus, the enhancement of both ferric ion reduction and lipid peroxidation may be due to oxalate increasing the solubility of the ferric ion. Increasing the oxalate concentration such that the oxalate/ferric ion ratio favored formation of the 2:1 and 3:1 complexes resulted in inhibition of iron reduction and lipid peroxidation. Our results confirm that hydroxyl radical formation occurs via the 2,5-DMBQ redox cycle.     

Effect of inducers of lipid peroxidation on the behavior of ciliated epithelial cells

Television microscope and original image treatment system were used for monitoring and recording the ciliary activity (beat frequency) of gill ciliated epithelia of the mussel Mytilus edulis (Bivalvia) and of the rat tracheal ciliated epithelia in response to the following prooxidants: H₂O₂, Fe⁺², Fe⁺² + ascorbic acid and NADP-H + ADP + Fe⁺². Mussel ciliated cells proved to be more sensitive to the influence of the prooxidants than rat cells. The reactions of ciliated epithelial cells of mollusks and rats to the inducers of lipid peroxidation were not similar to behavioral responses of these cells under the action of low-dose ionizing radiation.     

Effects of various iron supply on oxidative stress development and ferritin formation in the common ice plants

Mesembryanthemum crystallinum L. plants were grown from seeds in perlite. At the age of 4 weeks (juvenile plants) or 6 weeks (adult plants), they were transferred on nutrient media with different Fe³⁺ content brought in as Fe₂(SO₄)₃—EDTA complex (pH 6.0): control, iron deficit, and iron “excess”. Adult plants grown in media differing in iron content were subjected to salinity (300 mM NaCl) during the last 8 days of growth. Biochemical analyses were performed after plant fixation in liquid nitrogen; simultaneously, the samples for electron microscopy were taken. Different content of available Fe³⁺ in medium, especially under salinity conditions, changed sharply the content of chlorophyll and proline, the rate of lipid peroxidation, the level of H₂O₂, the activities of antioxidant enzymes in the leaves and roots, the number and sizes of plastoglobules, and ferritin formation in plastids. Joint action of salinity and iron deficit enhanced oxidative stress development, whereas iron excess hampered oxidative reaction development, reduced the rate of lipid peroxidation, and increased the chlorophyll content. At iron excess, plastoglobule lysis in plastids did not occur, their number and sizes increased, and ferritin deposits appeared, whereas the latter were absent at iron deficit.     

Paraquat and iron-dependent lipid peroxidation

The aim of this work was to study the effect of paraquat (P²⁺) on NADPH iron-dependent lipid peroxidation (basal peroxidation) either in the presence of NADPH or in the presence of NADPH-generating systems. When NADPH is present, P²⁺ potentiates NADPH iron-dependent lipid peroxidation, but use of NADPH-generating systems cancels this effect. This may be attributed to certain components in NADPH-generating systems such as glucose-6-phosphate and sodium isocitrate, which act as iron chelators. The binding of iron by these molecules facilitates its reduction and enhances its reactivity toward dioxygen molecules, leading to the formation of reactive species capable of initiating lipid peroxidation, such as Fe³⁺-O ₂ ⁻ . Under these conditions of rapid basal peroxidation, any additional reduction of iron(III) by a reduced form of P²⁺ (P^+.) has no apparent effect on the peroxidation itself, probably because the initial reaction between iron(II) and O₂ followed by initiation of the peroxidation are both rate-limiting steps in the process. Consequently, any alteration of the composition of the reacting mixture (e.g., buffers or the generating system) must be taken into consideration because the formation of new iron chelates can change the rate of basal peroxidation and will modify the effect of redoxcycling molecules.     

Inhibition of xanthine oxidase — xanthine — iron mediated lipid peroxidation by eugenol in liposomes

The effect of eugenol on xanthine oxidase (XO) xanthine(X)-Fe⁺³-ADP mediated lipid peroxidation was studied in liver microsomal lipid liposomes. Eugenol inhibited the lipid peroxidation in a dose dependent manner as assessed by formation of thiobarbituric acid reactive substances. When tested for its effect on XO activity per se, (by measuring uric acid formation) eugenol inhibited the enzyme to an extent of 85% at 10 µm concentration and hence formation of O₂ also However, the concentration of eugenol required for XO inhibition was more in presence of metal chelators such as EDTA, EGTA and DETAPAC, but not in presence of deferoxamine, ADP and citrate. The antiperoxidative effect of eugenol was about 35 times more and inhibition of XO was about 5 times higher as compared to the effect of allopurinol. Eugenol did not scavenge O₂ generated by phenazine methosulfate and NAD but inhibited propagation of peroxidation catalyzed by Fe²⁺ EDTA and lipid hydroperoxide containing liposomes. Eugenol inhibits XO-X-Fe⁺³ ADP mediated peroxidation by inhibiting the XO activity per se in addition to quenching various radical species. (Mol Cell Biochem 166: 65-71, 1997)     

Inactivation of the plasma membrane ATPase ofSchizosaccharomyces pombe by hydrogen peroxide and by the fenton reagent (Fe2+/H2O2): Nonradicalvs. Radical-induced oxidation

In the absence of added Fe²⁺, the ATPase activity of isolatedSchizosaccharomyces pombe plasma membranes (5–7 μmolP _i per mg protein per min) is moderately inhibited by H₂O₂ in a concentration-dependent manner. Sizable inactivation occurs only at 50–80 mmol/L H₂O₂. The process, probably a direct oxidative action of H₂O₂ on the enzyme, is not induced by the indigenous membrane-bound iron (19.3 nmol/mg membrane protein), is not affected by the radical scavengers mannitol and Tris, and involves a decrease of both theK _m of the enzyme for ATP and theV of ATP splitting. On exposing the membranes to the Fenton reagent (50 μmol/L Fe²⁺ +20 mmol/L H₂O₂), which causes a fast production of HO⁻ radicals, the ATPase is 50–60% inactivated and 90% of added Fe²⁺ is oxidized to Fe³⁺ within 1 min. The inactivation occurs only when Fe²⁺ is added before H₂O₂ and can thus bind to the membranes. The lack of effect of radical scavengers (mannitol, Tris) indicates that HO⁻ radicals produced in the bulk phase play no role in inactivation. Blockage of the inactivation by the iron chelator deferrioxamine implies that the process requires the presence of Fe²⁺ ions bound to binding sites on the enzyme molecules. Added catalase, which competes with Fe²⁺ for H₂O₂, slows down the inactivation but in some cases increases its total extent, probably due to the formation of the superoxide radical that gives rise to delayed HO⁻ production.     

Mechanisms of Saccharomyces Cerevisiae PMA1 H+-ATPase inactivation by Fe2+, H2O2 and Fenton reagents

Although considerably more oxidation-resistant than other P-type ATPases, the yeast PMA1 H⁺-ATPase of Saccharomyces cerevisiae SY4 secretory vesicles was inactivated by H₂O₂, Fe²⁺, Fe- and Cu-Fenton reagents. Inactivation by Fe²⁺ required the presence of oxygen and hence involved auto-oxidation of Fe²⁺ to Fe³⁺. The highest Fe^2- (100 μM) and H₂O₂ (100 mM) concentrations used produced about the same effect. Inactivation by the Fenton reagent depended more on Fe²⁺ content than on H₂O₂ concentration, occurred only when Fe²⁺ was added to the vesicles first and was only slightly reduced by scavengers (mannitol, Tris, NaN₃, DMSO) and by chelators (EDTA, EGTA, DTPA, BPDs, bipyridine, 1, 10-phenanthroline). Inactivation by Fe- and Cu- Fenton reagent was the same; the identical inactivation pattern found for both reagents under anaerobic conditions showed that both reagents act via OH^·. The lipid peroxidation blocker BHT prevented Fenton-induced rise in lipid peroxidation in both whole cells and in isolated membrane lipids but did not protect the H⁺-ATPase in secretory vesicles against inactivation. ATP partially protected the enzyme against peroxide and the Fenton reagent in a way resembling the protection it afforded against SH-specific agents. The results indicate that Fe²⁺ and the Fenton reagent act via metal-catalyzed oxidation at specific metal-binding sites, very probably SH-containing amino acid residues. Deferrioxamine, which prevents the redox cycling of Fe²⁺, blocked H⁺-ATPase inactivation by Fe²⁺ and the Fenton reagent but not that caused by H₂O₂, which therefore seems to involve a direct non-radical attack. Fe-Fenton reagent caused fragmentation of the H⁺-ATPase molecule, which, in Western blots, did not give rise to defined fragments bands but merely to smears.     

Protection of U937 cells from free radical damage by the macrophage synthesized antioxidant 7,8-dihydroneopterin

Interferon-γ stimulation of human macrophages causes the synthesis and release of neopterin and its reduced form 7,8-dihydroneopterin (7,8-NP). The purpose of this cellular response is undetermined but in vitro experiments suggests 7,8-NP is an antioxidant. We have found 7,8-NP can protect monocyte-like U937 cells from oxidative damage. 7,8-NP inhibited ferrous ion and hypochlorite mediated loss of cell viability. Fe⁺⁺ mediated lipid peroxidation was effectively inhibited by 7,8-NP, however no correlation was found between peroxide concentration and cell viability. Hypochlorite was scavenged by 7,8-NP, preventing the loss of cell viability. 7,8-NP was less effective in inhibiting H₂O₂-mediated loss of cell viability with significant inhibition only occurring at high 7,8-NP concentrations. Analysis of cellular protein hydrolysates showed none of the oxidants caused the formation of any protein bound DOPA or dityrosine but did show 7,8-NP prevented the loss of cellular tyrosine by HOCl. Our data suggests macrophages may synthesize 7,8-NP for antioxidant protection during inflammatory events in vivo.     

Decomposition of unsaturated phospholipid by iron-ADP-adriamycin co-ordination complex

The system, which contains NADPH, purified cytochrome P-450 reductase, and adriamycin, produces H₂O₂ and $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ in appreciable amounts with oxygen consumption and NADPH oxidation under aerobic conditions. Such an adriamycin-induced NADPH oxidation system, however, does not cause the decomposition of unsaturated fatty acids in microsomal phospholipid micelles, suggesting no direct participation of the active oxygen species and semiquinone radicals of adriamycin in lipid peroxidation. Adriamycin produces a co-ordination complex with Fe³⁺ and ADP, which, but no Fe³⁺-ADP complex, could be reduced by NADPH-cytochrome P-450 reductase at the expence of NADPH. The decomposition of unsaturated fatty acids in phospholipid micelles is achieved by the Fe³⁺-ADP-adriamycin complex and strikingly enhanced by enzymatically reduced iron-ADP-adriamycin complex.     

Oxygen consumption during the fenton-type reaction between hydrogen peroxide and a ferrous-chelate (Fe2+-DTPA)

The Fenton-type reaction between ferrous diethylenetriamine pentaacetic acid (Fe²⁺-DTPA, 50–200 μM) and H₂O₂ (20–1000 μM) in phosphate buffer at pH 7.0 results in consumption of dissolved oxygen. This observation differs from many prior reports that oxygen is liberated when more concentrated solutions of H₂O₂ are decomposed by iron salts. The rate and total quantity of oxygen consumed were dependent upon the concentrations of ferrous chelate, H₂O₂, and excess DTPA. Evidence is provided that both the ferrous-DTPA chelate and free DTPA can participate in the oxygen-consuming reactions. Oxygen was also consumed during the Fenton reaction between ferrous ions and H₂O₂ when DTPA and phosphate buffer were omitted. Under these conditions, oxygen evolution was observed at higher H₂O₂ concentrations (e.g., 400 μM). The consumption of oxygen during the Fenton-type reaction of an iron chelate at neutral pH may be relavant to events that take place in biologic systems.     

Cloning and characterization of the rat TIS11 gene

Pirfenidone (Pf), a new broad-spectrum anti-fibrotic agent, is known to offer protection against lung fibrosis in vivo in laboratory animals, and against mitogenesis and collagen formation by human lung fibroblasts in vitro. Because reactive oxygen species are thought to be involved in these events, we investigated the mechanism(s) by which Pf ameliorates oxidative stress and its effects on NADPH-dependent lipid peroxidation. Pf has been shown to cause inhibit NADPH-dependent lipid peroxidation in sheep liver microsomes in a dose-dependent manner. The concentration of Pf required to cause 50% inhibition of lipid peroxidation was ~ 6 mM. Pf was found to be ineffective as a superoxide radical scavenger. Pf was also ineffective in decomposing H₂O₂ and chelating iron. In deoxyribose degradation assays, Pf was a potent scavenger of hydroxyl radicals with a rate constant of 5.4 × 10⁹ M^-1 sec^-1. EPR spectroscopy in combination with spin trapping techniques, using a Fenton type reaction and DMPO as a spin-trapping agent, Pf scavenged hydroxyl radicals in a dose-dependent manner. The concentration of Pf required to inhibit 50% signal height was ~ 2.5 mM. Because iron was used in the Fenton reaction, the ability of Pf in chelating iron was verified in a fluorescent competitive assay using calcein as the fluorescent probe. Pf up to 10 mM concentration was ineffective in chelating either Fe²⁺ or Fe³⁺ in this system. We propose that Pf exerts its beneficial effects, at least in part, through its ability to scavenge toxic hydroxyl radicals.     

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

Analysis of lipid peroxidation mechanisms in human spermatozoa

The mechanisms by which ferrous ion promoters induce malondialdehyde generation by human spermatozoa have been investigated in order to provide a rational basis for the quantification and interpretation of lipid peroxidation assays. Incubation of human spermatozoa with a ferrous ion promoter in the presence of thiobarbituric acid (TBA) led to the generation of the bone fide malondialdehyde-TBA adduct. The importance of iron in the stimulation of lipid peroxidation was emphasized by the ability of Desferal* and EDTA to suppress malondialdehyde generation. Paradoxically, when the concentration of EDTA relative to iron was equimolar or greater, the suppression of malondialdehyde formation was accompanied by the generation of hydroxyl radicals. These results suggested that the addition of promoter did not effect the first-chain initiation of lipid peroxidation but favored an alternative mechanism involving the catalytic decomposition of pre-existing lipid peroxides. This conclusion was reinforced by the inability of reagents that would limit the formation (superoxide dismutase and/or catalase) or availability (mannitol, formate) of hydroxyl radicals, to influence malondialdehyde generation. While hydroxyl radicals were not directly involved in Fe²⁺-promoted malondialdehyde generation, the existence of significant correlations between reactive oxygen species production and the outcome of the TBA assay, suggested that Fenton chemistry might be important in the initiation of peroxidative damage. It is proposed that the impeded propagation of peroxidation initiated by Fenton or Haber Weiss reactions would lead to the accumulation of lipid peroxides in the spermatozoa and it is these peroxides that are induced to decompose during the Fe²⁺-promoted TBA assay, stimulating a lipoperoxidative chain reaction and malondialdehyde formation. © 1993 Wiley-Liss, Inc.     

Nadph-initiated cytochrome P450-dependent free iron-independent microsomal lipid peroxidation: Specific prevention by ascorbic acid

In this paper we demonstrate that ascorbic acid specifically prevents NADPH-initiated cytochrome P450 (P450)-mediated microsomal lipid peroxidation in the absence of free iron. Lipid peroxidation has been evidenced by the formations of conjugated dienes, lipid hydroperoxide and malondialdehyde. Other scavengers of reactive oxygen species including superoxide dismutase, catalase, glutathione, -tocopherol, uric acid, thiourea, mannitol, histidine, -carotene and probucol are ineffective to prevent the NADPH-initiated P450-mediated free iron-independent microsomal lipid peroxidation. Using a reconstituted system comprised of purified NADPH-P450 reductase, P450 and isolated microsomal lipid or pure L--phosphatidylcholine diarachidoyl, a mechanism has been proposed for the iron-independent microsomal lipid peroxidation and its prevention by ascorbic acid. It is proposed that the perferryl moiety P450 Fe³⁺. O₂ initiates lipid peroxidation by abstracting methylene hydrogen from polyunsaturated lipid to form lipid radical, which then combines with oxygen to produce the chain propagating peroxyl radical for subsequent formation of lipid peroxides. Apparently, ascorbic acid prevents initiation of lipid peroxidation by interacting with P450 Fe³⁺. O₂. (Mol Cell Biochem 166: 35-44, 1997)     

Coordinating subdomains of ferritin protein cages with catalysis and biomineralization viewed from the C 4 cage axes

Integrated ferritin protein cage function is the reversible synthesis of protein-caged, solid Fe₂O₃·H₂O minerals from Fe²⁺ for metabolic iron concentrates and oxidant protection; biomineral order differs in different ferritin proteins. The conserved 432 geometric symmetry of ferritin protein cages parallels the subunit dimer, trimer, and tetramer interfaces, and coincides with function at several cage axes. Multiple subdomains distributed in the self-assembling ferritin nanocages have functional relationships to cage symmetry such as Fe²⁺ transport though ion channels (threefold symmetry), biomineral nucleation/order (fourfold symmetry), and mineral dissolution (threefold symmetry) studied in ferritin variants. On the basis of the effects of natural or synthetic subunit dimer cross-links, cage subunit dimers (twofold symmetry) influence iron oxidation and mineral dissolution. 2Fe²⁺/O₂ catalysis in ferritin occurs in single subunits, but with cooperativity (n = 3) that is possibly related to the structure/function of the ion channels, which are constructed from segments of three subunits. Here, we study 2Fe²⁺ + O₂ protein catalysis (diferric peroxo formation) and dissolution of ferritin Fe₂O₃·H₂O biominerals in variants with altered subunit interfaces for trimers (ion channels), E130I, and external dimer surfaces (E88A) as controls, and altered tetramer subunit interfaces (L165I and H169F). The results extend observations on the functional importance of structure at ferritin protein twofold and threefold cage axes to show function at ferritin fourfold cage axes. Here, conserved amino acids facilitate dissolution of ferritin-protein-caged iron biominerals. Biological and nanotechnological uses of ferritin protein cage fourfold symmetry and solid-state mineral properties remain largely unexplored.     

Effects of lipid peroxidation on adrenal microsomal monooxygenases

Incubation of guinea pig adrenal microsomes with 10^?6 M ferrous (Fe²⁺) ion and adrenal cytosol initiated high levels of lipid peroxidation as measured by the production of malonaldehyde. Cytosol or Fe²⁺ alone had little effect on microsomal malonaldehyde formation. When microsomes were incubated in the presence of Fe²⁺ and cytosol, malonaldehyde levels continued to increase for at least 60 min. Accompanying the lipid peroxidation was a decline in adrenal microsomal monooxygenase activities. The rates of metabolism of xenobiotics (benzphetamine demethylase, benzo[α]pyrene hydroxylase) as well as steroids (21-hydroxylation) decreased as malonaldehyde levels increased. In addition, cytochrome P-450 levels, NADPH- and NADH-cytochrome c reductase activities, and substrate interactions with cytochrome(s) P-450 decreased as lipid peroxidation progressed. Inhibition of lipid peroxidation by increasing microsomal protein concentrations during the incubation period prevented the changes in microsomal metabolism. Malonaldehyde had no direct effects on adrenal microsomal enzyme activities. The results indicate that lipid peroxidation may have significant effects on adrenocortical function, diminishing the capacity for both xenobiotic and steroid metabolism.     

Peroxidation of linoleic acid during the oxidation of phenols by fungal laccase

A system comprising laccase and a suitable phenol such as 4-hydroxybenzoic acid (HBA) or synthetic lignin (DHP) exhaustively peroxidized linoleic acid in acetate buffer. The presence of phenols in lignin was essential since an exhaustively methylated preparation of the same lignin did not support peroxidation. The peroxidation rate was greatly enhanced by Mn²⁺, which was oxidized to Mn³⁺ by laccase/HBA, whereas H₂O₂ inhibited strongly due to rapid reduction of Mn³⁺ by H₂O₂ with concomitant formation of O₂. When acetate was replaced by Mn³⁺–chelating oxalate or malonate, there was no change in peroxidation rates in the absence of Mn²⁺, whereas strong inhibition was observed in the presence of Mn²⁺. In case of malonate part of the inhibition was due to H₂O₂ formation as a result of Mn³⁺ reduction by malonate. These findings suggest that laccase may contribute to fungal lipid peroxidation in vivo thus expanding its role in the biodegradation of lignin and other recalcitrant aromatic compounds.     

Competitive Inhibition of Ferrous Iron Oxidation by Thiobacillus ferrooxidans by Increasing Concentrations of Cells

The oxidation of ferrous iron (Fe²⁺) to ferric iron (Fe³⁺) with dioxygen (O₂) by various strains of Thiobacillus ferrooxidans was studied by measuring the rate of O₂ consumption at various Fe²⁺ concentrations and cell concentrations. The apparent K_m values for Fe²⁺ remained constant at different cell concentrations of laboratory strains ATCC 13661 and ATCC 19859 but increased with increasing cell concentrations of mine isolates SM-4 and SM-5. The latter results are explained by the competitive inhibition of the Fe²⁺-binding site of a cell by other cells in the reaction mixture. Possible mechanisms involving cell surface properties are discussed.