Increased oxidative stress brought on by pro-inflammatory cytokines in neurodegenerative processes and the protective role of nitrone-based free radical traps.

Nitrone-based free radical traps (NFTs) have been shown to be protective in several neurodegenerative models. Our research has strongly implicated that: A) several neurodegenerative conditions exhibit increased levels of pro-inflammatory cytokines which consequently result in increased levels of oxidative stress and B) that NFTs act in part by suppressing oxidative stress through suppression of the action of the cytokine cascade. Acquired Immune Deficiency Syndrome (AIDS) dementia complex (ADC) is one of several conditions where the data collected helped to develop these concepts. Novel observations include demonstration that IL-1beta acts on cultured brain glia cells to invoke protein nitration and oxidative stress and that low levels of PBN (alpha-phenyl tert-butyl nitrone) inhibit this effect. We interpret these data as indicating that PBN prevents IL-1beta mediated peroxynitrite formation. Additionally, we have found that the AIDS viral envelope protein gp120 upregulates mRNA for the cytokines TNF alpha and TNF beta in rat neonatal brain, and that PBN prevents this. Western blots of protein extracts showed upregulation of inducible nitric oxide synthase (iNOS) in gp120 treated neonatal rat brains, and that PBN prevented induction of this enzyme as well. These observations underscore the general concept that PBN inhibits the induction of genes which produce neurotoxic products, one of which is peroxynitrite formed by the reaction of nitric oxide with superoxide, and may act also by inhibiting the induction of cytokines which mediate pro-inflammatory conditions in the brain.

The presence of two distinct 8-oxoguanine repair enzymes in human cells: their potential complementary roles in preventing mutation.

8-Oxoguanine (8-oxoG), induced by reactive oxygen species (ROS) and ionizing radiation, is arguably the most important mutagenic lesion in DNA. This oxidized base, because of its mispairing with A, induces GC-->TA transversion mutations often observed spontaneously in tumor cells. The human cDNA encoding the repair enzyme 8-oxoG-DNA glycosylase (OGG-1) has recently been cloned, however, its activity was never detected in cells. Here we show that the apparent lack of this activity could be due to the presence of an 8-oxoG-specific DNA binding protein. Moreover, we demonstrate the presence of two antigenically distinct OGG activities with an identical reaction mechanism in human cell (HeLa) extracts. The 38 kDa OGG-1, identical to the cloned enzyme, cleaves 8-oxoG when paired with cytosine, thymine and guanine but not adenine in DNA. In contrast, the newly discovered 36 kDa OGG-2 prefers 8-oxoG paired with G and A. We propose that OGG-1 and OGG-2 have distinct antimutagenic functions in vivo . OGG-1 prevents mutation by removing 8-oxoG formed in DNA in situ and paired with C, while OGG-2 removes 8-oxoG that is incorporated opposite A in DNA from ROS-induced 8-oxodGTP. We predict that OGG-2 specifically removes such 8-oxoG residues only from the nascent strand, possibly by utilizing the same mechanism as the DNA mismatch repair pathway.

Potential mechanisms of photodynamic inactivation of virus by methylene blue. I. RNA-protein crosslinks and other oxidative lesions in Q beta bacteriophage.

A spectrum of oxidative lesions was observed in a bacteriophage-based model system that is very sensitive to the photodynamic activity of selected dyes. When suspensions of the intact bacteriophage Q beta were exposed to methylene blue plus light (MB + L), inactivating events, or "hits" occurred that were oxygen-dependent and that were associated with the formation of several specific lesions: (1) carbonyl moieties on proteins, (2) 8-oxo-7,8-dihydroguanine (8-oxoGua), and (3) single-strand breaks (ssb) in the RNA genome and (4) RNA-protein crosslinks. Formation of carbonyl groups associated with protein in the Q beta phage preparation correlated positively with photoinactivation of the phage with increasing doses of either of the sensitizers MB or rose bengal. Strand breaks in the Q beta genomic RNA were observable at high MB concentrations but appeared not to be significant at the lower concentrations of MB, as full-length Q beta RNA was observable well beyond the 99% inactivation point in MB dosage. It was shown that the number of 8-oxoGua lesions were unlikely to be sufficient to account for the number of lethal events. Following exposure to MB + L, crosslink formation between Q beta RNA and protein was observed by virtue of the location of RNA at the interface of phenol-aqueous extractions of phage suspensions. A significant increase over background of RNA-protein complexes (including full-length Q beta RNA) was observed at the lowest concentration of MB tested (0.5 microM), which corresponded roughly to an average of 2 lethal hits per phage or approximately 13% survival compared to the zero MB control (100% survival). Due to its close correlation with Q beta inactivation and its expected lethality, RNA-protein crosslink formation may be important as an inactivating lesion in bacteriophage Q beta following MB + L exposure.

8-Hydroxydeoxyguanosine in DNA from TPA-stimulated human granulocytes.

8-Hydroxydeoxyguanosine (8-OHdG) is now widely used as a sensitive marker of oxidative damage to DNA. When human granulocytes are stimulated with TPA, they release a large quantity of reactive oxygen species (superoxide, hydrogen peroxide) which might be expected to generate hydroxyl radicals (OH.) which in turn could produce 8-OHdG in the DNA. There had been considerable debate as to whether OH. is detectable in stimulated granulocytes; most workers now agree that none can be detected, unless exogenous iron is added. An earlier report had described that 8-OHdG (a marker of OH.) was increased in the DNA of TPA-stimulated, compared to control, granulocytes. We have repeated this experiment and have been unable to reproduce this finding. We conclude that the amount of 8-OHdG produced in the DNA of TPA-stimulated human granulocytes is indistinguishable from that seen in control (unstimulated) cells (less than one 8-OHdG/10(5) dG).

Superoxide dismutase and Fenton chemistry. Reaction of ferric-EDTA complex and ferric-bipyridyl complex with hydrogen peroxide without the apparent formation of iron(II).

A ferric-EDTA complex, prepared directly from FeCl3 or from an oxidized ferrous salt, reacts with H2O2 to form hydroxyl radicals (.OH), which degrade deoxyribose and benzoate with the release of thiobarbituric acid-reactive material, hydroxylate benzoate to form fluorescent dihydroxy products and react with 5,5-dimethylpyrrolidine N-oxide (DMPO) to form a DMPO-OH adduct. Degradation of deoxyribose and benzoate and the hydroxylation of benzoate are substantially inhibited by superoxide dismutase and .OH-radical scavengers such as formate, thiourea and mannitol. Inhibition by the enzyme superoxide dismutase implies that the reduction of the ferric-EDTA complex for participation in the Fenton reaction is superoxide-(O2.-)-dependent, and not H2O2-dependent as frequently implied. When ferric-bipyridyl complex at a molar ratio of 1:4 is substituted for ferric-EDTA complex (molar ratio 1:1) and the same experiments are conducted, oxidant damage is low and deoxyribose and benzoate degradation were poorly if at all inhibited by superoxide dismutase and .OH-radical scavengers. Benzoate hydroxylation, although weak, was, however, more effectively inhibited by superoxide dismutase and .OH-radical scavengers, implicating some role for .OH. The iron-bipyridyl complex had available iron-binding capacity and therefore would not allow iron to remain bound to buffer or detector molecules. Most .OH radicals produced by the iron-bipyridyl complex and H2O2 are likely to damage the bipyridyl molecules first, with few reacting in free solution with the detector molecules. Deoxyribose and benzoate degradation appeared to be mediated by an oxidant species not typical of .OH, and species such as the ferryl ion-bipyridyl complex may have contributed to the damage observed.

ADP-iron as a Fenton reactant: radical reactions detected by spin trapping, hydrogen abstraction, and aromatic hydroxylation.

A mixture of ADP, ferrous ions, and hydrogen peroxide (H2O2) generates hydroxyl radicals (OH) that attack the spin trap DMPO (5,5-dimethyl-pyrollidine-N-oxide) to yield the hydroxyl free radical spin-adduct, degrade deoxyribose and benzoate with the release of thiobarbituric acid-reactive material, and hydroxylate benzoate to give fluorescent products. Inhibition studies, with scavengers of the OH radical, suggest that the behavior of iron-ADP in the reaction is complicated by the formation of ternary complexes with certain scavengers and detector molecules. In addition, iron-ADP reacting with H2O2 appears to release a substantial number of OH radicals free into solution. During the generation of OH radicals the ADP molecule was, as expected, damaged by the iron bound to it. Damage to the iron ligand in this way is not normally monitored in reaction systems that use specific detector molecules for OH radical damage. Under certain reaction conditions the ligand may be the major recipient of OH radical damage thereby leading to the incorrect assumption that the iron ligand is a poor Fenton reactant.

Methylene blue plus light mediates 8-hydroxy 2'-deoxyguanosine formation in DNA preferentially over strand breakage.

Methylene blue (MB) plus light, in the presence of oxygen, mediates formation of 8-hydroxyguanine in DNA. The yield of 8-hydroxyguanine may be as much as from 2 to 4% of the guanines present. The results presented here show that treatment of supercoiled plasmid DNA with methylene blue plus light causes single-stranded nicks. However, single-stranded nicking occurs approximately 17-fold less frequently than does formation of 8-hydroxyguanine. The nicking rate is reduced in the presence of Mg ion but is not prevented by inhibitors of the iron-catalyzed Fenton reaction or by scavengers of hydroxyl free radicals. Extensive exposure of DNA to light in the presence of MB produces no detectable thiobarbital reactive material thus implicating that single strand nicking does not occur by hydroxyl free radical attack on deoxyribose. Formation of 8-hydroxyguanine is apparently not dependent upon intercalative binding of MB to DNA, since it is formed in polydeoxyguanylic acid.