The bioactivity of neuronal-derived nitric oxide in aging and neurodegeneration: Switching signaling to degeneration.

The small and diffusible free radical nitric oxide (•NO) has fascinated biological and medical scientists since it was promoted from atmospheric air pollutant to biological ubiquitous signaling molecule. Its unique physical chemical properties expand beyond its radical nature to include fast diffusion in aqueous and lipid environments and selective reactivity in a biological setting determined by bioavailability and reaction rate constants with biomolecules. In the brain, •NO is recognized as a key player in numerous physiological processes ranging from neurotransmission/neuromodulation to neurovascular coupling and immune response. Furthermore, changes in its bioactivity are central to the molecular pathways associated with brain aging and neurodegeneration. The understanding of •NO bioactivity in the brain, however, requires the knowledge of its concentration dynamics with high spatial and temporal resolution upon stimulation of its synthesis. Here we revise our current understanding of the role of neuronal-derived •NO in brain physiology, aging and degeneration, focused on changes in the extracellular concentration dynamics of this free radical and the regulation of bioenergetic metabolism and neurovascular coupling.

p21WAF1 regulates anchorage-independent growth of HCT116 colon carcinoma cells via E-cadherin expression.

The cyclin-dependent kinase inhibitor p21WAF1 has been characterized as an important effector of the tumor suppressor p53 and has been linked to various growth-regulatory processes. To identify a potential role of p21 in anchorage-dependent growth control, we analyzed a pair of HCT116 human colon carcinoma cell lines that differed only in their p21 status. We found that during suspension culture, HCT116 cells (which contain wildtype p53 and p21) continued to proliferate and formed compact multicellular spheroids (MCSs). In contrast, HCT116 cells engineered to lack functional p21 (HCTp21-/-) were unable to form MCSs in suspension culture, ceased proliferation, and eventually died through apoptosis. The parental HCT116 cells underwent the same fate when treated with hyaluronidase, indicating that cell-cell contact might be required for survival in suspension culture. We established that E-cadherin was induced in HCT116 but not in HCTp21-/- cells and accounted for the formation of MCSs. Forced expression of E-cadherin or p21 in HCTp21-/- cells restored the ability to form MCSs and to grow independently of anchorage. Moreover, HCTp21-/- cells exhibited a severely reduced transformed phenotype and demonstrated greatly enhanced chemosensitivity in suspension culture. Thus, our results link an important regulator of the cell cycle machinery to the expression of a cell-cell adhesion molecule involved in tumor formation. Because our results indicate that loss of p21 severely impairs the ability of HCT cells to grow independently of anchorage, it may not be coincidental that inactivating mutations of this gene are very rarely found in tumor cells.

Tobacco Smoke-Induced Hepatic Injury with Steatosis, Inflammation, and Impairments in Insulin and Insulin-Like Growth Factor Signaling.

BACKGROUND: Alcoholic liver disease (ALD) is associated with impairments in hepatic insulin and insulin-like growth factor (IGF) signaling through cell growth, survival, and metabolic pathways. Since not all heavy drinkers develop ALD, co-factors may be important. Epidemiologic data indicate that most heavy drinkers smoke tobacco and experimental data revealed that low-level nitrosamine exposures, including those from tobacco, can cause steatohepatitis with hepatic insulin/IGF resistance and exacerbate ALD. We hypothesize that cigarette smoke (CS) exposures also cause liver injury with impaired hepatic insulin/IGF signaling, and thereby contribute to ALD. METHODS: Adult male A/J mice were exposed to air for 8 weeks (A8), CS for 4 (CS4) or 8 (CS8) weeks, or CS for 8 weeks with 2 weeks recovery (CS8+R). RESULTS: CS exposures caused progressive liver injury with disruption of the normal hepatic chord architecture, lobular inflammation, apoptosis or necrosis, micro-steatosis, sinusoidal dilatation, and nuclear pleomorphism. Histopathological liver injury scores increased significantly from A8 to CS4 and then further to CS8 (P<0.0001). The mean histological grade was also higher in CS8+R relative to A8 (P<0.0001) but lower than in CS4, reflecting partial resolution of injury by CS withdrawal. CS exposures impaired insulin and IGF-1 signaling through IRS-1, Akt, GSK-3ß, and PRAS40. Livers from CS8+R mice had normalized or elevated levels of insulin receptor, pYpY-Insulin-R, 312S-IRS-1, 473S-Akt, S9-GSK-3ß, and pT246-PRAS40 relative to A8, CS4, or CS8, reflecting partial recovery. CONCLUSION: CS-mediated liver injury and steatohepatitis with impairments in insulin/IGF signalling are reminiscent of the findings in ALD. Therefore, CS exposures (either first or second-hand) may serve as a co-factor in ALD. The persistence of several abnormalities following CS exposure cessation suggests that some aspects of CS-mediated hepatic metabolic dysfunction are not readily reversible.

Role of p53 in aziridinylbenzoquinone-induced p21waf1 expression.

Quinones are the second largest family of anticancer drugs clinically used in the United States. However, their exact mode of action at the cellular and molecular level is not completely understood. We have shown earlier that the quinone 3,6-diaziridinyl-1,4-benzoquinone (DZQ) leads to the increased expression of p21waf1/cip1/sdi1 protein, an inhibitor of cyclin-dependent kinases. Because p21 has been established as an important negative regulator of the cell cycle, we further investigated the molecular basis of p21 induction by DZQ. Here we report that the induction of p21 by DZQ is regulated at the transcriptional level, and requires the activation of p53, a tumor suppressor protein. In cells that lack functional p53 protein, DZQ-mediated p21 induction is greatly diminished. However, the introduction of a wild type p53 gene into p53-negative cells restores the strong DZQ-inducibility of p21. Restoration of wild type p53 status in HL60 myeloid leukemia cells significantly increases the cells' sensitivity to the cytotoxic effects of DZQ. Thus, our results indicate that the p53-p21 pathway may play a central role in mediating the gene-regulatory and cytotoxic effects of aziridinylbenzoquinones.

The role of mitochondrial glutathione in DNA base oxidation.

The objective of this study was to elucidate the role of mitochondrial GSH in the reactions leading to mitochondrial DNA oxidative damage in terms of 8-hydroxy-desoxyguanosine (8-HOdG) accumulation. With this purpose, tightly coupled mitochondria depleted of matrix GSH were used and the effects of H2O2 (generated during the oxidation of substrates) on 8-HOdG levels were investigated. Mitochondrial integrity, assessed by O2 uptake, respiratory control and P/O ratios, was conserved upon depletion of GSH up to 95%. The rates of H2O2 production linked to the oxidation of endogenous substrates by control and GSH-depleted mitochondria were similar. Succinate (in the absence or presence of antimycin A) enhanced the rate H2O2 production to a similar extent in both control and GSH-depleted mitochondria. These rates of H2O2 production accounted for 1.5-2.5% of the rate of O2 uptake. The levels of 8-HOdG in GSH-depleted mitochondria were 35-50% lower than those in control mitochondria, when measured at different H2O2 production rates. Conversely, in experiments carried out with calf thymus DNA with different Cu/Fe content, GSH increased 1.4-2.4-fold the accumulation of 8-HOdG. These values were further enhanced (44-50%) by superoxide dismutase and decreased by catalase. The lower levels of 8-HOdG in GSH-depleted mitochondria and the higher levels in GSH-supplemented calf thymus DNA suggest a role for the non-protein thiol in the reactions leading to mtDNA oxidative damage. These findings are interpreted in terms of the redox transitions involving O2, GSH, and metal catalysts bound to DNA. A mechanism is proposed by which GSH plays a critical role in the reduction of DNA-Cu complexes and decays by free radical pathways kinetically regulated by superoxide dismutase.

Analysis of the kinetic mechanism of halophilic NADP-dependent glutamate dehydrogenase.

The amination of 2-oxoglutarate catalyzed by NADP-specific glutamate dehydrogenase (EC 1.4.1.4, L-glutamate:NADP+ oxidoreductase (deaminating)) from Halobacterium halobium has been analyzed by initial rate, graphical analysis, and product and competitive inhibition studies. Initial rate and graphical analysis reveal that a B term (representing 2-oxoglutarate) is not statistically necessary for an initial rate equation. However, the absence of a B term does not distinguish between ordered and random binding of NADPH and ammonia. The patterns of product inhibition by NADP+ and L-glutamate, and competitive inhibition by hydroxylamine and succinate permit deduction of the kinetic mechanism as ordered, with NADPH, 2-oxoglutarate and ammonia added in that order, and L-glutamate release preceding NADP+ release.

Kinetic mechanism of Halobacterium halobium NAD+-glutamate dehydrogenase.

The kinetic mechanism of Halobacterium halobium NAD+-glutamate dehydrogenase (EC 1.4.1.3) has been investigated at pH 9.0, 3 M NaCl and 40 degrees C in both directions, by initial rate and inhibition studies. The results of the initial rate studies indicate that the mechanism is sequential with respect to substrate addition. The inhibition patterns obtained with halophilic NAD+-glutamate dehydrogenase are not consistent with a simple ordered mechanism without modification. They can, however, be reconciled with this type of mechanism by postulating an appropriate abortive complex.

Electronically excited state generation during the reaction of p-benzoquinone with H2O2. Relation to product formation: 2-OH- and 2,3-epoxy-p-benzoquinone. The effect of glutathione.

The reaction between H2O2 and p-benzoquinone proceeds with consumption of both reactants with second order rate constants of 1.66- and 0.77 M-1S-1, respectively. The process is mainly supported by oxygen addition reactions to the quinone resulting in the formation of both 2,3-epoxy-p-benzoquinone and 2-OH-p-benzoquinone. The former product accumulates in the assay mixture without participating in further reactions. The formation of the latter product implies free radical intermediates such as 2-OH-p-benzosemiquinone anion, which supports the generation of electronically excited states upon its oxidation by H2O2, presumably as part of an organic Fenton reaction. The relaxation of the excited state is accompanied by photoemission at 485-530 nm. Glutathione was found to counteract the oxidative aspects of the reaction between p-benzoquinone and H2O2 by a series of processes involving (a) a rapid reductive addition to the quinone with formation of a substituted p-benzohydroquinone; (b) an effective quenching of photoemission, which might be attributed to the deactivation of the excited state by the p-benzohydroquinone-glutathione adduct, and (c) the decomposition of the formed 2,3-epoxy-p-benzoquinone, also by reductive cleavage of the epoxide ring.

Heme protein radicals: formation, fate, and biological consequences.

The oxidation of myoglobin by H2O2 yields ferrylmyoglobin, which contains two oxidizing equivalents: the oxoferryl complex and an amino acid radical. This study examines the electron paramagnetic resonance (EPR) properties of the resulting amino acid radicals and their inherent kinetic features at [H2O2]/[protein] ratios close to physiological conditions (i.e., < or = 1). The EPR spectrum obtained with continuous flow at room temperature consisted of a composite of three signals: a low intensity signal and two high intensity signals. The former had a g-value of 2.014, contributed 10-15% to the overall spectrum and was ascribed to a peroxyl radical. Of the two high intensity signals, one consisted of a six-line spectrum (g = 2.0048) that contributed approximately 17-19% to the overall signal; hyperfine splitting constants to ring protons permitted to identify this signal as a tyrosyl radical. The other high intensity signal (with similar g-value and underlying that of the tyrosyl radical) was ascribed to an aromatic amino acid upon comparison with the EPR characteristics for radicals in aromatic amino acid-containing peptides. Analysis of these data in connection with amino acid analysis and the EPR spectra obtained under similar conditions with another hemoprotein, hemoglobin, allowed to suggest a mechanism for the formation of the protein radicals in myoglobin. The aromatic amino acid radical was observed to be relatively long lived in close proximity to the heme iron. Hence, it is likely that this is the first site of protein radical; reduction of the oxoferryl complex by Tyr (FeIV=O + Tyr-OH + H+ --> FeIII + H2O + Tyr-O.)--and alternatively by other amino acids--leads to the subsequent formation of other amino acid radicals within an electron-transfer process throughout the protein. This view suggests that the protein radical(s) is highly delocalized within the globin moiety in a dynamic process encompassing electron tunneling through the backbone chain or H-bonds and leading to the formation of secondary radicals.

Oxidation of adrenaline by ferrylmyoglobin.

The oxidation of adrenaline by ferrylmyoglobin, the product formed by the oxidation of myoglobin with H2O2, was examined by absorption, fluorescence, and EPR spectroscopy in terms of the formation of intermediate free radicals and stable molecular products and the binding of adrenaline oxidation products to the apoprotein. The reaction of adrenaline with ferrylmyoglobin resulted in reduction of the hemoprotein to metmyoglobin and consumption of adrenaline. Quantification of metmyoglobin formed per adrenaline yielded a ratio of 1.66. The reaction was found first order on adrenaline concentration and second order on ferrylmyoglobin concentration. This, together with the above ratio, suggested a mechanism by which two oxoferryl moieties (ferrylmyoglobin) were reduced by adrenaline yielding metmyoglobin and the o-semiquinone state of adrenaline. The decay of the o-semiquinone to adrenochrome was confirmed by an increase in absorbance at 485 nm. The product was nonfluorescent; alkalinization of the reaction mixture resulted in a strong fluorescence at 540 nm ascribed to 3,5,6-trihydroxyindol or adrenolutin. Hence, adrenochrome and its alkali-catalyzed product, adrenolutin, are the major molecular products formed during the oxidation of adrenaline by ferrylmyoglobin. Semiquinones formed during the adrenaline/ferrylmyoglobin interaction were detected by EPR, spin stabilizing these species with Mg2+. The six-line EPR spectrum observed (aN=4.5 G, aN(CH3)=5.1, and a2H=0.91; g=2.0040) may be assigned to the semiquinone forms of adrenochrome and/or adrenolutin or a composite of these species. The intensity of the EPR signal increased with time and its subsequent decay followed a second-order kinetics as inferred by the proportionality of the square of the EPR line intensity with H2O2 concentration. Heme destruction and lysine loss, inherent in the reaction of metmyoglobin with H2O2, were prevented 80 and 34% by adrenaline, respectively. The low protection exerted by adrenaline against lysine loss was possibly due to the formation of Schiff bases between the epsilon-NH2 group of lysine and the o-quinone oxidation product(s) of adrenaline. The yield of Schiff base formation was 20-25%. The autoxidation of adrenaline at physiological pH is extremely slow or nonexistent. These data provide a rationale for the primary oxidation of adrenaline by the pseudoperoxidatic activity of ferrylmyoglobin and suggest implications of the free radicals thereby formed for the oxidative damage in reperfusion injury.

Estimation of H2O2 gradients across biomembranes.

When cells are exposed to an external source of H2O2, the rapid enzymatic consumption of H2O2 inside the cell provides the driving force for the formation of the gradient across the plasma and other subcellular membranes. By using the concepts of enzyme latency, the following gradients - formed after a few seconds following the exposure to H2O2 - were estimated in Jurkat T-cells: [H2O2](cytosol)/[H2O2](peroxisomes)=3; [H2O2](extracellular)/[H2O2](cytosol)=7. The procedure presented in this work can easily be applied to other cell lines and provides a quantitative framework to interpret the data obtained when cells are exposed to an external source of H2O2.

1,4-Reductive addition of glutathione to quinone epoxides. Mechanistic studies with h.p.l.c. with electrochemical detection under anaerobic and aerobic conditions. Evaluation of chemical reactivity in terms of autoxidation reactions.

The nucleophilic addition of GSH to quinonoid compounds, characterized as a 1,4-reductive addition of the Michael type, was studied with p-benzoquinone- and 1,4-naphthoquinone epoxides with different degree of methyl substitution. Identification and evaluation of molecular products from the above reaction were assessed by h.p.l.c. with either reductive or oxidative electrochemical detection, based on the redox properties retained in the molecular products formed. It was found that the degree of methyl substitution of the quinone epoxide, from either the 1,4-naphthoquinone- or p-benzoquinone epoxide series, determined their rate of reaction with GSH. The reductive addition implied the rearrangement of the quinone structure with opening of the epoxide ring yielding as the primary product a hydroxy-glutathionyl substituted adduct of either p-benzohydroquinone or 1,4-naphthohydroquinone. The primary product undergoes elimination reactions and redox transitions which bring about a number of secondary molecular products. The distribution pattern of the latter depends on the degree of methyl substitution of the quinone epoxide studied and on the concentration of O2 in the solution. The occurrence of the hydroxy-substituent in position alpha, adjacent to the carbonyl group, enhances the autoxidation properties of the compound resulting in an augmented O2 consumption and H2O2 production. Therefore, it could be expected that the chemical reactivity of the products originating from the thiol-mediated nucleophilic addition to quinone epoxides would be of toxicological interest.

Redox and addition chemistry of quinoid compounds and its biological implications.

The overall biological activity of quinones is a function of the physico-chemical properties of these compounds, which manifest themselves in a critical bimolecular reaction with bioconstituents. Attempts have been made to characterize this bimolecular reaction as a function of the redox properties of quinones in relation to hydrophobic or hydrophilic environments. The inborn physico-chemical properties of quinones are discussed on the basis of their reduction potential and dissociation constants, as well as the effect of environmental factors on these properties. Emphasis is given on the effect of methyl-, methoxy-, hydroxy-, and glutathionyl substituents on the reduction potential of quinones and the subsequent electron transfer processes. The redox chemistry of quinoid compounds is surveyed in terms of a) reactions involving only electron transfer, as those accomplished during the enzymic reduction of quinones and the non-enzymic interaction with redox couples generating semiquinones, and b) nucleophilic addition reactions. The addition of nucleophiles, entailing either oxidation or reduction of the quinone, are exemplified in reactions with oxygen- or sulfur nucleophiles, respectively. The former yields quinone epoxides, whereas the latter yields thioether-hydroquinone adducts as primary molecular products. The subsequent chemistry of these products is examined in terms of enzymic reduction, autoxidation, cross-oxidation, disproportionation, and free radical interactions. The detailed chemical mechanisms by which quinoid compounds exert cytotoxic, mutagenic and carcinogenic effects are considered individually in relation to redox cycling, alterations of thiol balance and Ca++ homeostasis, and covalent binding.

Cellular titration of apoptosis with steady state concentrations of H(2)O(2): submicromolar levels of H(2)O(2) induce apoptosis through Fenton chemistry independent of the cellular thiol state.

Apoptosis was studied under conditions that mimic the steady state of H(2)O(2) in vivo. This is at variance with previous studies involving a bolus addition of H(2)O(2), a procedure that disrupts the cellular homeostasis. The results allowed us to define three phases for H(2)O(2)-induced apoptosis in Jurkat T-cells with reference to cytosolic steady state concentrations of H(2)O(2) [(H(2)O(2))(ss)]: (H(2)O(2))(ss) values below 0.7 microM elicited no effects; (H(2)O(2))(ss) approximately 0.7-3 microM induced apoptosis; and (H(2)O(2))(ss) > 3 microM yielded no additional apoptosis and a gradual shift towards necrosis as the mode of cell death were observed. H(2)O(2)-induced apoptosis was not affected by either BCNU, an inhibitor of glutathione reductase, or diamide, a compound that reacts both with low-molecular weight and protein thiols, or selenols. Glutathione depletion, accomplished by incubating cells either with buthionine sulfoximine or in cystine-free medium, rendered cells more sensitive to H(2)O(2)-induced apoptosis, but did not change the threshold and saturating concentrations of H(2)O(2) that induced apoptosis. Two unrelated metal chelators, desferrioxamine and dipyridyl, strongly protected against H(2)O(2)-induced apoptosis. It may be concluded that, under conditions of H(2)O(2) delivery that mimic in vivo situations, the oxidative event that triggers the induction of apoptosis by H(2)O(2) is a Fenton-type reaction and is independent of the thiol or selenium states of the cell.

The reaction of ascorbic acid with different heme iron redox states of myoglobin. Antioxidant and prooxidant aspects.

The interaction of ascorbate with different heme iron redox states of myoglobin (ferrylmyoglobin, FeIV = O; metmyoglobin, FeIII; and oxymyoglobin, FeIIO2) was examined by e.s.r. and absorption spectroscopy. The reaction of ascorbate with ferryl- or met-myoglobin resulted in ascorbyl radical production. The interaction of ascorbate with oxymyoglobin proceeded with formation of ascorbyl radical, hydrogen peroxide, and an overall oxidation of oxymyoglobin to metmyoglobin. The latter reaction proceeded via an oxoferryl complex intermediate-corresponding to ferrylmyoglobin and identified by treatment of the reaction mixture with Na2S. These observations are consistent with a concerted electron transfer mechanism, whereby the two electrons required for the reduction of oxygen to hydrogen peroxide are donated by ascorbic acid and the heme iron. The antioxidant and prooxidant aspects of these redox transitions are discussed in terms of their kinetic properties.

Pulse radiolysis study of the reactivity of Trolox C phenoxyl radical with superoxide anion.

The reaction between the phenoxyl radical of Trolox C, a water-soluble vitamin E analogue, and superoxide anion radical was examined by using the pulse radiolysis technique. The results indicate that the Trolox C phenoxyl radical may undergo a rapid one-electron transfer from superoxide radical [k = (4.5 +/- 0.5) x 10(8) M-1.S-1] to its reduced form. This finding indicates that superoxide radical might play a role in the repair of vitamin e phenoxyl radical.

Reactivity of metmyoglobin towards phospholipid hydroperoxides.

Ferrylmyoglobin, the high oxidation state of myoglobin analogous to compound II of peroxidases, promotes the peroxidation of palmitoyl-linoleyl-phosphatidylcholine (PLPC) large unilamellar vesicles. This was associated with oxygen consumption and a slow conversion of ferrylmyoglobin to metmyoglobin. The time course of oxygen consumption was characterized by the occurrence of a lag phase, which could be overcome by the addition of sodium deoxycholate to the reaction mixture. The rate of conversion of ferrylmyoglobin to metmyoglobin was slower than that of oxygen consumption, and there was not stoichiometric correlation between both events. These findings suggest that the observed oxygen consumption linked to lipid peroxidation is supported by a peroxidatic activity encompassed by the ferrylmyoglobin<==>metmyoglobin transition as well as free radical propagation reactions. Incubation of metmyoglobin with PLPC vesicles containing 3% hydroperoxide resulted in oxygen consumption, the time course of which was devoid of the lag phase observed with hydroperoxide-free unilamellar lipid vesicles. The incubation of metmyoglobin with peroxide-containing PLPC vesicles or with equimolar amounts of lipid hydroperoxide was not associated with Soret or visible absorption spectral changes of metmyoglobin, which could be ascribed to its conversion to ferrylmyoglobin. Treatment of the metmyoglobin/lipid hydroperoxide mixtures with Na2S did not lead to the formation of the sulfheme protein derivative, which can be considered as a fingerprint for the occurrence of ferrylmyoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione-dependent reduction of peroxides during ferryl- and met-myoglobin interconversion: a potential protective mechanism in muscle.

Met-myoglobin is oxidized both by H2O2 and other hydroperoxides to a species with a higher iron valency state and the spectral characteristics of ferryl-myoglobin. Glutathione (GSH) reduces the latter species back to met-myoglobin with parallel oxidation to its disulfide (GSSG) but cannot reduce met-myoglobin to ferrous myoglobin. Under aerobic conditions, the GSH-mediated reduction of ferry-myoglobin is associated with O2 consumption and amounts of GSSG are formed far in excess over that of the peroxide added. Under anaerobic conditions, this ratio is close to unity. These results are interpreted in terms of a one-electron redox process involving the reduction of ferryl-myoglobin to met-myoglobin and the one-electron oxidation of GSH to its thiyl radical. Further reactions of thiyl radicals are influenced by the presence of oxygen which will be the determining factor in the ratio H2O2 added/GSSG formed. It is suggested that, when oxygen is limiting, myoglobin may serve as a protector of muscle cells against peroxides and other oxidants.

Mitochondrial free radical generation, oxidative stress, and aging.

Mitochondria have been described as "the powerhouses of the cell" because they link the energy-releasing activities of electron transport and proton pumping with the energy conserving process of oxidative phosphorylation, to harness the value of foods in the form of ATP. Such energetic processes are not without dangers, however, and the electron transport chain has proved to be somewhat "leaky." Such side reactions of the mitochondrial electron transport chain with molecular oxygen directly generate the superoxide anion radical (O2*-), which dismutates to form hydrogen peroxide (H2O2), which can further react to form the hydroxyl radical (HO*). In addition to these toxic electron transport chain reactions of the inner mitochondrial membrane, the mitochondrial outer membrane enzyme monoamine oxidase catalyzes the oxidative deamination of biogenic amines and is a quantitatively large source of H2O2 that contributes to an increase in the steady state concentrations of reactive species within both the mitochondrial matrix and cytosol. In this article we review the mitochondrial rates of production and steady state levels of these reactive oxygen species. Reactive oxygen species generated by mitochondria, or from other sites within or outside the cell, cause damage to mitochondrial components and initiate degradative processes. Such toxic reactions contribute significantly to the aging process and form the central dogma of "The Free Radical Theory of Aging." In this article we review current understandings of mitochondrial DNA, RNA, and protein modifications by oxidative stress and the enzymatic removal of oxidatively damaged products by nucleases and proteases. The possible contributions of mitochondrial oxidative polynucleotide and protein turnover to apoptosis and aging are explored.

One-electron transfer reactions of diquat radical to different reduction intermediates of oxygen. Formation of hydroxyl radical and electronically excited states.

The one-electron transfer activation of DQ++ by microsomal fractions comprises an aerobic phase and an anaerobic phase. The aerobic phase is characterized by O2 consumption, formation of electronically excited states with main emission below 600 nm, and H2O2 formation. The anaerobic phase is characterized by H2O2 consumption, DQ+ accumulation, HO. formation, and also electronically excited state formation with main emission beyond 600 nm. Superoxide dismutase abolishes the photoemission during the aerobic phase, whereas it has no effect on the photoemission originating during the anaerobic phase. The hydroxylation products of the aromatic compound salicylate, mainly 2,3- and 2,5-dihydroxybenzoic acids--indicative of the occurrence of HO.-, were detected by h.p.l.c. with oxidative electrochemical detection during the anaerobic phase, but not during the aerobic phase. Neither H2O2 consumption nor HO. are prevented by desferrioxamine. These experimental observations are interpreted on the grounds of two main electron-transfer reactions of DQ.+: under aerobic conditions, two one-electron transfer steps to molecular O2 and O2.- to yield H2O2. Under anaerobic conditions, one-electron transfer step to contaminating iron or any ferrioxamine formed to a ferrous complex which can support a Fenton-like reduction of H2O2 with formation of HO.. The toxicological relevance for the occurrence of such reactions is also discussed in terms of the formation of electronically excited states.