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E Cadenas

Publications and source records attributed to E Cadenas.

At least 37 records · Page 2Linked to original sources
PubMed1998

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.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Apoproteins↗
PubMed1998

Extracellular activation of fluorinated aziridinylbenzoquinone in HT29 cells EPR studies.

The plasma membrane of HT29 human colon carcinoma cells was characterized by EPR spectroscopy as the site for redox activation of 3,6-difluoro-2,5-bis(aziridinyl)-1,4-benzoquinone (F-DZQ). Supplementation of HT29 cells with F-DZQ yielded an EPR signal ascribed to the semiquinone species; the hyperfine splitting constants of the 11-line spectrum were 1.4 and 1.35 G for aN and aF, respectively. The intensity of the EPR signal was inhibited competitively by potassium ferricyanide, a compound which has no access to the intracellular milieu and used to evaluate transmembrane NADH-ferricyanide reductase activity. The extracellular localization of the signal was confirmed by using chromium trioxalate, a membrane-impermeant spin-broadening agent, which abolished in a concentration-dependent manner the semiquinone signal originating from the metabolism of F-DZQ by HT29 cells. The intensity of the semiquinone signal was decreased by agents which block sulfhydryl groups upon alkylation, fluorodinitrobenzene and p-chloromercuribenzoate, presumably acting on plasma membrane dehydrogenases. Other flavin dehydrogenase inhibitors, such as allopurinol, deprenyl or clorgyline, and D-arginine or NG-methyl-L-arginine did not affect the EPR signal. Conversely, the intensity of the semiquinone signal was increased upon supplementation of HT29 cells with glucose and insulin, which may enhance the intracellular levels of electron donors for the transplasma membrane dehydrogenase activity. The extracellular semiquinone signal was abolished by superoxide dismutase by a mechanism implying displacement of the equilibrium of the autoxidation reaction. Formation of oxygen-centered radicals during this redox activity was evaluated by EPR in conjunction with the spin trap 4-POBN. A composite signal consisting of the spin adducts of methyl, hydroxyl and superoxide radicals was observed (the former arising from hydroxyl radical attack on the quinone solvent, dimethylsulfoxide). The formation of these spin adducts was abolished by superoxide dismutase and their detection became impossible in the presence of the line broadening agent chromiun trioxalate, thus indicating their extracellular formation and localization, respectively. The occurrence of a redox site at the plasma membrane of HT29 cells for the activation of this halogenated aziridinylbenzoquinone is discussed in terms of its significance for intracellular processes and a build-up of oxyradicals in the extracellular milieu.

Alkylating Agents↗
PubMed1998

Anticancer quinones induce pRb-preventable G2/M cell cycle arrest and apoptosis.

Reactive oxygen species generated during the metabolism of the antitumor quinone 3,6-diaziridinyl-1,4-benzoquinone (DZQ) in human colonic carcinoma HCT116 cells lead to the induction of p21 (WAF1, Cip1, or sdi1), an upstream regulator of the retinoblastoma gene product pRb involved G1 cell cycle control. We here demonstrate that the cell cycle was arrested in G2/M phase following supplementation with DZQ of human osteosarcoma Saos-2 cells (lacking both p53 and pRb) and HCT116 cells. DZQ also induced p21 and apoptosis in Saos-2 cells. The transfection of the Rb gene into Saos-2 cells did not alter the level of p21 induction, but changed cell cycle arrest into G1 phase and prevented apoptosis. These findings suggest that quinones may lead to a p53-independent and pRb-preventable G2/M arrest and apoptosis, which correlate with p21 induction.

Antineoplastic Agents↗
PubMed1998

Reactions of halogen-substituted aziridinylbenzoquinones with glutathione. Formation of diglutathionyl conjugates and semiquinones.

The reaction between glutathione and 2,5-diaziridinyl-1,4-benzoquinones bearing halogen substituents at C3 and C6 was examined in terms of the formation of glutathionyl-quinone conjugates and semiquinones by HPLC with UV detection, mass spectroscopy and EPR. The reactivity of the halogen atoms toward sulfur substitution is the primary reaction leading to the formation of mono- and di-glutathionyl-substituted quinones. The relative formation of these conjugates depended on the GSH/quinone molar ratios. At GSH/quinone molar ratios below unity, the products observed were the reduced form of the parent quinone, a dithioether derivative and GSSG. Disulfide formation accounted for 60-68% of total GSH consumed. EPR analysis of these reaction mixtures showed a 5-line spectrum (1:2:3:2:1 relative intensities) with 2 equivalent N (aN = 1.98 G) and assigned to the semiquinone form of dichloro- diaziridinylbenzoquinone. Semiquinone quantification by double integration of the EPR signals and interpolation with an adequate standard revealed that the amount of semiquinone formed per GSH consumed was 0.98. At GSH/quinone molar ratios above unity (4, 10 and 100 molar excess of GSH) a pattern of products emerged consisting of 3,6-diglutathionyl quinones with two, one and no aziridinyl moieties, identified by mass spectral analysis. EPR studies revealed that these compounds were minor components of a composite EPR spectrum (a 3-line signal with 1:1:1 relative intensities, 1 equivalent N (aN = 1.73 G) and 1 H (aH = 1.45 G) or a 3-line signal with 1:2:1 relative intensities and 2 equivalent H (aH = 1.4 G). These minor components were assigned to the diglutathionyl conjugates bearing one- or no aziridinyl moiety, respectively. The major component in the EPR signal showed a 3-line spectrum (1:1:1 relative intensity) with 1 equivalent N (aN = 1.7 G) and a g shift of -0.96 G. This spectrum was assigned to a triglutathionyl conjugate of a monoaziridinylbenzoquinone. This major component was also observed when GSH/quinone mixtures were incubated with the two-electron transfer flavoprotein NAD(P)H:quinone oxidoreductase. The semiquinone signals were abolished by superoxide dismutase. In the presence of catalase, the contribution of these components to the overall EPR spectrum was equal. These data are discussed in terms of the one-electron transfer steps encompassed by thiol oxidation and semiquinone formation and the two-electron transfers inherent in sulfur substitution and aziridinyl group loss.

Antineoplastic Agents↗
PubMed1998

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.

Amino Acids↗
PubMed1998

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart.

Isolated rat heart perfused with 1.5-7.5 microM NO solutions or bradykinin, which activates endothelial NO synthase, showed a dose-dependent decrease in myocardial O2 uptake from 3.2 +/- 0.3 to 1.6 +/- 0.1 (7.5 microM NO, n = 18, P < 0.05) and to 1.2 +/- 0.1 microM O2.min-1.g tissue-1 (10 microM bradykinin, n = 10, P < 0.05). Perfused NO concentrations correlated with an induced release of hydrogen peroxide (H2O2) in the effluent (r = 0.99, P < 0.01). NO markedly decreased the O2 uptake of isolated rat heart mitochondria (50% inhibition at 0.4 microM NO, r = 0.99, P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b and cytochrome c, which accounts for the effects in O2 uptake and H2O2 release. Most NO was bound to myoglobin; this fact is consistent with NO steady-state concentrations of 0.1-0.3 microM, which affect mitochondria. In the intact heart, finely adjusted NO concentrations regulate mitochondrial O2 uptake and superoxide anion production (reflected by H2O2), which in turn contributes to the physiological clearance of NO through peroxynitrite formation.

Animals↗
PubMed1998

The lag phase.

Peroxidation of lipids in membranes and lipoproteins proceeds through the classical free radical sequence encompassing initiation, propagation, and termination phases which are expressed by a lag phase in which little oxidation occurs, followed by a rapid increase in autocatalysis by chain-propagating intermediates and, finally, a decrease in the rate of oxidation. The lag phase is lengthened by preventive or chain-breaking antioxidants, which scavenge the initiation reaction or intercept the chain-carrying species. Hence, the lag phase in lipid peroxidation processes reflects the antioxidant status of membranes and lipoproteins and, as a corollary, their resistance to oxidation. A large number of lipid peroxidation studies with different membranes attest to the complex free radical network underlying this process. The type of initiator and the steady-state level of oxygen are important factors that affect differently the rates of the individual steps of peroxidation. Equally complex are the factors that influence the lag phase preceding the oxidation of LDL. Lipid peroxyl radicals play a key role in the dynamics of lipid peroxidation: on the one hand, the lag phase is best defined for chain-breaking compounds able to reduce peroxyl radicals; on the other hand, the overall time course of lipid peroxidation is largely influenced by the rate constants for propagation reactions and termination involving peroxyl radical recombination.

Animals↗
PubMed1997

The role of NAD(P)H:quinone oxidoreductase in quinone-mediated p21 induction in human colon carcinoma cells.

This study examines the role of NAD(P)H:quinone acceptor oxidoreductase (NQOR) (EC 1.6.99.2) in the metabolism of aziridinylbenzoquinones and the ensuing formation of reactive oxygen species in the induction of the cell cycle inhibitor p21 (WAF1, Cip1, or sdi1) in human colon carcinoma cells. The aziridinylbenzoquinones used were 2,5-diaziridinyl-1,4-benzoquinone (DZQ) and 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone (AZQ). The cell lines used in this study, BE and HT29 human colon carcinoma cell lines, are devoid of and overexpress NQOR activity, respectively. The rate of reduction of the above quinones in BE cells proceeded at similar rates (approximately 170 nmol/min/ mg protein) and, expectedly, it was not affected by the NQOR inhibitor, dicumarol. The metabolism of DZQ in HT29 cells was largely accomplished by NQOR (approximately 94%), whereas that of AZQ was accomplished by dicumarol-insensitive reductases. The metabolism of DZQ in HT29 cells was accompanied by H2O2 formation, which was approximately 10-fold higher than that ensuing from the activation of AZQ. In agreement with these data, the production of H2O2 during the activation of DZQ by purified NQOR was approximately 10-fold higher than that of AZQ. The formation of H2O2 during the metabolism of aziridinylbenzoquinones in BE cells was 24- to 57-fold lower than that in HT29 cells. At variance with HT29 cells, H2O2 formation by BE cells was insensitive to the catalase inhibitor sodium azide. The bioactivation of AZQ and DZQ in BE cells yielded O2.- and HO. as detected by spin trapping/EPR, the intensity of the former adduct being approximately 2-fold higher than that of the latter. These signals were insensitive to dicumarol. The metabolism of DZQ in HT29 cells yielded mainly HO. and a modest contribution of O2.- (ratio HO./O2.- approximately 10), whereas that of AZQ yielded a HO./O2.- approximately 2. The effect of dicumarol on the free radical pattern obtained during DZQ metabolism resulted in a strong inhibition (80%) of HO. production and a substantial increase of O2.- generation. The metabolism of DZQ and AZQ in BE cells was associated with a significant increase of p21 mRNA levels; the former quinone was approximately 2-fold more efficient than the latter. DZQ metabolism in HT29 cells led to an increase of p21 mRNA levels 15-fold higher than that observed with AZQ activation. Dicumarol did not inhibit p21 induction associated with the metabolism of DZQ in the NQOR-deficient BE cells, whereas the inhibitor decreased p21 induction in HT29 cells by approximately 30%. This modest inhibition is likely due to the low concentration of dicumarol used, which did not affect p21 constitutive levels in control experiments carried out in the absence of the quinone. p21 induction in HT29 cells was also inhibited by DTPA, a metal chelator, and N-acetylcysteine, a potent cellular anti-oxidant, suggesting that HO. may serve as an ultimate mediator for the induction. It may be surmised that the higher efficiency of DZQ in p21 induction may be related to its efficient metabolism by NQOR in HT29 cells and the associated high level of reactive oxygen species. The role of reactive oxygen species in p21 induction was further assessed upon supplementation of cells with H2O2:p21 induction in BE cells was 4-fold higher than that in HT29 cells. These findings suggest that assessment of the role of NQOR and reactive oxygen species in p21 induction requires careful consideration of the cell genotype.

Acetylcysteine↗
PubMed1996

Induction of p21 mediated by reactive oxygen species formed during the metabolism of aziridinylbenzoquinones by HCT116 cells.

Aziridinylbenzoquinones are a group of antitumor agents that elicit cytotoxicity by generating either alkylating intermediates or reactive oxygen species. The mechanism of toxicity may not always, however, involve profound damage of cellular constituents, but may involve a cytostatic effect through interference with the cell cycle. In this context, we have examined the induction of the cell cycle inhibitor p21 (WAF1, CIP1, or sdi1), whose overexpression suppresses the growth of various tumor cells, in human tumor cells metabolizing 3,6-diaziridinyl-1,4-benzoquinone (DZQ) and its C2,C5-substituted derivatives: 2,5-bis-(carboethoxyamino) (AZQ) and 2, 5-bis-2(-hydroxyethylamino) (BZQ). Both DZQ and AZQ were effectively activated by HCT116 human colonic carcinoma cells; the activation of the former involved largely a dicoumarol-sensitive activity, whereas that of the latter appeared to be accomplished primarily by one-electron transfer reductases. BZQ was not a substrate for the dicoumarol-sensitive enzyme in HCT116 cells. Cellular activation of the first two quinones was associated with formation of oxygen-centered radicals as detected by EPR in conjunction with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide. The redox transitions of DZQ involved hydroxyl radical formation and were strongly inhibited by catalase, whereas those of AZQ showed a strong superoxide anion component sensitive to superoxide dismutase. These signals were suppressed by N-acetylcysteine with concomitant production of a thiyl radical adduct. This suggests an effective electron transfer between the thiol and free radicals formed during the activation of these quinones. DZQ and AZQ induced significantly the expression of p21 in HCT116 cells, but a 10-fold higher concentration of AZQ was required to achieve the level of induction elicited by DZQ. BZQ had little effect on p21 expression. p21 induction at both mRNA and protein levels correlated with the inhibition of either cyclin-dependent kinase activity or cell proliferation. p21 induction elicited by the above quinones was inhibited by N-acetylcysteine, whereas the non-sulfur analog, N-acetylalanine, was without effect. Catalase and superoxide dismutase did not effect p21 induction by aziridinylbenzoquinones in HCT116 cells, thus suggesting that extracellular sources of oxygen radicals generated by plasma membrane reductases have no influence in the expression of this gene. Hydrogen peroxide, a product of quinone redox cycling, elicited an increase of p21 mRNA levels in HCT116 and K562 human chronic myelogenous leukemia cells. The latter lacks p53, one of the activators of p21 transcription, thus suggesting that p21 expression can be accomplished in a p53-independent manner in these cells. This study suggests that p21 induction is mediated by an increase in the cellular steady-state concentration of oxygen radicals and that the greater effectiveness in p21 induction by DZQ may be related to its efficient metabolism by NAD(P)H:quinone oxidoreductase activity in HCT116 cells.

Acetylcysteine↗
PubMed1996

The metabolism of tyramine by monoamine oxidase A/B causes oxidative damage to mitochondrial DNA.

Monoamine oxidases A/B (EC 1.4.3.4, MAO), flavoenzymes located on the outer mitochondrial membrane, catalyze the oxidative deamination of biogenic amines, such as dopamine, serotonin, and norepinephrine. In this study, we examined whether the H2O2 formed during the two-electron oxidation of tyramine [4-(2-aminoethyl)phenol] (a substrate for monoamine oxidases A/B) may contribute to the intramitochondrial steady-state concentration of H2O2 ([H2O2]ss) and, thus, be involved in the oxidative impairment of mitochondrial matrix components. Supplementation of intact, coupled rat brain mitochondria with benzylamine, beta-phenylethylamine, or tyramine showed initial rates of H2O2 production ranging from 0.4- to 1.6 nmol H2O2/min/mg protein. ESR analysis of the oxidative deamination of tyramine by intact rat brain mitochondria revealed the formation of hydroxyl (HO.) and carbon-centered radical adducts--the latter probably originating by the (HO.-)-mediated oxidation of mannitol. The signals were substantially enhanced upon addition of FeSO4 and were abolished by catalase. The intramitochondrial [H2O2]ss calculated in terms of glutathione peroxidase activity during the metabolism of tyramine was 48-fold higher (7.71 +/- 0.25 x 10(-7) M) than that obtained during the oxidation of succinate via complex II in the presence of antimycin A (1.64 +/- 0.2 x 10(-8) M). Oxidative damage to the brain mtDNA was assessed by single strand breakage. The ratio of nicked DNA for the preparations treated with tyramine and those without the amine was 1.5 +/- 0.29 (n = 4), 2.12 +/- 0.28 (n = 8, P < or = 0.05), and 3.12 +/- 0.69 (n = 3, P < or = 0.05) at 15, 30, and 60 min, respectively . Preincubation of mitochondria with tranylcypromine (trans-2-phenylcyclopropylamine), an inhibitor to MAO A/B, abolished mtDNA oxidative damage. Catalase inhibited mtDNA strand breakage by approximately 60%. Incubation of intact, coupled rat brain mitochondria with chlorodinitrobenzene (CDNB) depleted mitochondrial GSH by 72%. Tyramine-dependent damage of mtDNA was decreased by 68% in CDNB-treated mitochondria (with 28% remaining GSH). The [H2O2]ss was slightly increased in CDNB-treated mitochondria: 1.38- and 1.28-fold increase during the oxidation of succinate in the presence of antimycin A and during the oxidation of tyramine, respectively. These results suggest that the H2O2 generated during the MAO-catalyzed oxidation of biogenic amines and possibly certain neurotransmitters at the outer mitochondrial membrane contributes to the intramitochondrial [H2O2]ss and may cause oxidative damage to mtDNA. This is effected by the intramitochondrial concentration of GSH and might have potential implications for aging and neurodegenerative processes.

Animals↗
PubMed1995

Hydroxyl radical generation during mitochondrial electron transfer and the formation of 8-hydroxydesoxyguanosine in mitochondrial DNA.

Production of hydroxyl radicals (HO.) by substrate-supplemented beef heart submitochondrial particles was studied by electron paramagnetic resonance in conjunction with the spin trap 5,5'-dimethyl-1-pirroline-N-oxide (DMPO). Supplementation of submitochondrial particles with NADH or succinate in the presence of antimycin resulted in the formation hydroxyl-, alpha-hydroxyethyl-, and methyl radical adducts. The latter two adducts were derived from HO. attack of ethanol or dimethyl sulfoxide (DMSO), respectively, the solvents used for the inhibitors of the respiratory chain. These ESR signals were slightly increased by superoxide dismutase and abolished by catalase. Further support for the production of HO. during mitochondrial electron transfer was furnished by kinetic competition experiments with DMSO as the HO. scavenger. This approach yielded a kappa SCAVENGER/kappa DMPO value of 1.7, in agreement with a competitive spin trapping of free HO. using DMSO as a scavenger. The scission of H2O2 to HO. requires consideration of a Fenton chemistry, i.e., the participation of metals or redox active metal pools in mitochondria to drive this reaction. The effect of several metal chelators on the formation of both HO. and H2O2 was examined. Bathophenantroline, bathocuproine, and desferrioxamine decreased the DMPO-HO. signal and increased accumulation of H2O2. Conversely, EDTA or diethylenetriaminepentaacetic acid substantially increased the DMPO-HO. signal intensity and decreased H2O2 accumulation. These different results were rationalized in terms of the reduction potential of the redox couples involved, i.e., that of the ligated metal and those encompassed in the one-electron reduction of superoxide radical and of hydrogen peroxide. The formation of 8-hydroxydesoxyguanosine in mitochondrial DNA was examined under experimental conditions in which H2O2 production by isolated mitochondria was enhanced. The formation of 8-hydroxydesoxyguanosine increased with increasing rates of H2O2 formation. The biological significance of H2O2 and HO. formation during mitochondrial electron transfer is discussed in terms of oxidative damage of mitochondrial DNA and the implications for mitochondrial functions and aging.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1995

Enzymic- and thiol-mediated activation of halogen-substituted diaziridinylbenzoquinones: redox transitions of the semiquinone and semiquinone-thioether species.

Activation of 2,5-diaziridinyl-1,4-benzoquinones bearing halogen (Cl, Br, or F) substituents at C3 and C6 by NADPH-cytochrome P450 reductase and glutathione nucleophilic substitution was examined in terms of free radical production and DNA strand scission. A semiquinone species was observed by direct ESR in aerobic conditions during: (a) NADPH-cytochrome P450 reductase-catalyzed reduction of the above quinones. (b) The interaction of these quinones with GSH entailing primarily reactivity of halogen substituents toward sulfur substitution. (c) NADPH-cytochrome P450 reductase-catalyzed activation of products resulting from the quinone/GSH interaction. The semiquinone ESR signal observed during enzymic catalysis was suppressed by superoxide dismutase and was not affected by catalase. ESR studies in conjunction with the spin trapping technique on the autoxidation of the semiquinones formed by the above reaction pathways indicated the formation of superoxide radicals. In addition, thiyl radicals were formed during the reactions following glutathione necleophilic substitution of the above quinones. The ESR signals of both superoxide and thiyl radicals were abolished by superoxide dismutase. No hydroxyl radicals were formed in solution during the redox transitions of these halogen-containing diaziridinylbenzoquinones. Bioreductive activation of these compounds via NADPH-cytochrome P450 reductase or sulfur nucleophilic substitution was associated with the formation of DNA strand breaks. This process was substantially inhibited (74-86%) by superoxide dismutase and to a lesser extent (23-31%) by catalase. It is suggested that DNA strand breakage proceeds in a manner entailing a semiquinone-dependent reduction of metal-ligands bound at the DNA surface and leading to site-specific, hydroxyl radical production.

Animals↗