Intracellular oxidation of hydroethidine: Compartmentalization and cytotoxicity of oxidation products

Hydroethidine (HE) is a blue fluorescent dye that is intracellularly converted into red-emitting products on two-electron oxidation. One of these products, namely 2-hydroxyethidium, is formed as the result of HE superoxide anion-specific oxidation, and so HE is widely used for the detection of superoxide in cells and tissues. In our experiments we exploited three cell lines of different origin: K562 (human leukemia cells), A431 (human epidermoid carcinoma cells), and SCE2304 (human mesenchymal stem cells derived from endometrium). Using fluorescent microscopy and flow cytometry analysis, we showed that HE intracellular oxidation products accumulate mostly in the cell mitochondria. This accumulation provokes gradual depolarization of mitochondrial membrane, affects oxygen consumption rate in HE-treated cells, and causes cellular apoptosis in the case of high HE concentrations and/or long cell incubations with HE, as well as a high rate of HE oxidation in cells exposed to some stimuli.     

Protein oxidation and aging

Organisms are constantly exposed to various forms of reactive oxygen species (ROS) that lead to oxidation of proteins, nucleic acids, and lipids. Protein oxidation can involve cleavage of the polypeptide chain, modification of amino acid side chains, and conversion of the protein to derivatives that are highly sensitive to proteolytic degradation. Unlike other types of modification (except cysteine oxidation), oxidation of methionine residues to methionine sulfoxide is reversible; thus, cyclic oxidation and reduction of methionine residues leads to consumption of ROS and thereby increases the resistance of proteins to oxidation. The importance of protein oxidation in aging is supported by the observation that levels of oxidized proteins increase with animal age. The age-related accumulation of oxidized proteins may reflect age-related increases in rates of ROS generation, decreases in antioxidant activities, or losses in the capacity to degrade oxidized proteins.     

Protein oxidation and aging

Organisms are constantly exposed to various forms of reactive oxygen species (ROS) that lead to oxidation of proteins, nucleic acids, and lipids. Protein oxidation can involve cleavage of the polypeptide chain, modification of amino acid side chains, and conversion of the protein to derivatives that are highly sensitive to proteolytic degradation. Unlike other types of modification (except cysteine oxidation), oxidation of methionine residues to methionine sulfoxide is reversible; thus, cyclic oxidation and reduction of methionine residues leads to consumption of ROS and thereby increases the resistance of proteins to oxidation. The importance of protein oxidation in aging is supported by the observation that levels of oxidized proteins increase with animal age. The age-related accumulation of oxidized proteins may reflect age-related increases in rates of ROS generation, decreases in antioxidant activities, or losses in the capacity to degrade oxidized proteins.     

Protein oxidation and proteolysis

One of the hallmarks of chronic or severe oxidative stress is the accumulation of oxidized proteins, which tend to form high-molecular-weight aggregates. The major proteolytic system responsible for the removal of oxidized cytosolic and nuclear proteins is the proteasome. This complicated proteolytic system contains a core proteasomal form (20S proteasome) and several regulators. All of these components are affected by oxidative stress to various degrees. The ATP-stimulated 26S proteasome is sensitive to oxidative stress, whereas the 20S form seems to be more resistant. The nuclear proteasome selectively degrades oxidatively damaged histones in the nuclei of mammalian cells, where it is activated and regulated by automodified PARP-1 after oxidative challenge. In this brief review we highlight the proteolysis and its regulatory effects during oxidative stress.     

Methionine oxidation and aging

It is well established that many amino acid residues of proteins are susceptible to oxidation by various forms of reactive oxygen species (ROS), and that oxidatively modified proteins accumulate during aging, oxidative stress, and in a number of age-related diseases. Methionine residues and cysteine residues of proteins are particularly sensitive to oxidation by ROS. However, unlike oxidation of other amino acid residues, the oxidation of these sulfur amino acids is reversible. Oxidation of methionine residues leads to the formation of both R- and S-stereoisomers of methionine sulfoxide (MetO) and most cells contain stereospecific methionine sulfoxide reductases (Msr's) that catalyze the thioredoxin-dependent reduction of MetO residues back to methionine residues. We summarize here results of studies, by many workers, showing that the MetO content of proteins increases with age in a number of different aging models, including replicative senescence and erythrocyte aging, but not in mouse tissues during aging. The change in levels of MetO may reflect alterations in any one or more of many different mechanisms, including (i) an increase in the rate of ROS generation; (ii) a decrease in the antioxidant capacity; (iii) a decrease in proteolytic activities that preferentially degrade oxidized proteins; or (iv) a decrease in the ability to convert MetO residues back to Met residues, due either to a direct loss of Msr enzyme levels or indirectly to a loss in the availability of the reducing equivalents (thioredoxin, thioredoxin reductase, NADPH generation) involved. The importance of Msr activity is highlighted by the fact that aging is associated with a loss of Msr activities in a number of animal tissues, and mutations in mice leading to a decrease in the Msr levels lead to a decrease in the maximum life span, whereas overexpression of Msr leads to a dramatic increase in the maximum life span.     

Formaldehyde oxidation and methanogenesis

Formaldehyde oxidation by cell-free extracts of Methanobacterium thermoautotrophicum was shown to drive methanogenesis from CH3-S-coenzyme M or HCHO under a nonreductive atmosphere of N2. Under N2 when HCHO was the sole source of carbon and reducing equivalents in the reaction, it underwent oxidation and reduction events (disproportionation), the sum of the reactions being 3 HCHO + H2O----CH4 + 2 HCOO - + 2H+. This reaction predicts a CH4/HCHO ratio of 1/3, which is in agreement with the experimental finding of 1/2.9. In extracts of the mesophilic methanogen Methanococcus voltae and the extreme thermophile Methanococcus jannaschii , which exhibited formate dehydrogenase activity, the CH4/HCHO ratio was 1/2. NADPH stimulated methane formation from HCHO under N2. An unidentified, oxygen-labile cofactor, the formaldehyde activation factor, present in boiled-cell extract was discovered. Methanopterin , an oxygen-stable molecule, also substituted for boiled-cell extract.     

Protein oxidation and turnover

All biomacromolecules are faced with oxidative stress. Oxidation of a protein molecule always induces inactivation of the molecule and introduces a tag to that molecule. These modified protein molecules are prone to degradation in vivo by the proteasome system. Coupling of protein modification and degradation of chemically modified proteins is one of the normal protein turnover pathways in vivo. We call this a 'chemical apoptosis' process, which is one of the early manifestations of programmed cell death. Impairment of the proteasome system leads to accumulation of modified nonfunctional proteins or 'aged proteins' that might cause various clinical syndromes including cataractogenesis, premature aging, neurological degeneration and rheumatoid disease. The metal-catalyzed oxidation of biomacromolecules provides an excellent artificial aging system in vitro. The system is very useful in the characterization of structure and function relationships of proteins (enzymes), especially in those containing metal binding domain(s), because the oxidation is always followed by an affinity cleavage at the metal binding site(s) that allows easy identification and further characterization.     

Protein and DNA oxidation in spinal injury: neurofilaments--an oxidation target

This study measured the time courses of protein and DNA oxidation following spinal cord injury (SCI) in rats and characterized oxidative degradation of proteins. Protein carbonyl content-a marker of protein oxidation-significantly increased at 3-9 h postinjury and the ratio 8-hydroxy-2-deoxyguanosine/deoxyguanosine-an indicator of DNA oxidation-was significantly higher at 3-6 h postinjury in the injured cords than in the sham controls. This suggests that oxidative modification of proteins and DNA contributes to secondary damage in SCI. Densities of selected bands on coomassie-stained gels indicated that most proteins were degraded. Neurofilament protein (NFP) was particularly evaluated immunohistochemically; its light chain (NFP-68) was gradually degraded in nerve fibers, neuron bodies, and large dendrites following SCI. A mixture of Mn (III) tetrakis (4-benzoic acid) porphyrin (10 mg/kg)-a novel SOD mimetic-and nitro-L-arginine (1 mg/kg)-an inhibitor of nitric oxide synthase-injected intraperitoneally, increased NFP-68 immunoreactivity and the numbers of NFP-positive nerve fibers post-SCI, correlating NFP degradation in SCI to free radical-triggered oxidative damage for the first time. Therefore, blockage of protein and DNA oxidation in the secondary injury stage may improve long-term recovery-important information for development of the SCI therapies.     

Peroxynitrite-mediated oxidation of dichlorodihydrofluorescein and dihydrorhodamine

The oxidations of dichlorodihydrofluorescein and dihydrorhodamine by peroxynitrite are zero-order in the indicator between pH 3 and 10. The yield of the oxidized products, dichlorofluorescein and rhodamine, significantly increased at pH values>7, and the maximal molar yields were 0.47 +/- 0.04 mol rhodamine and 0.54 +/- 0.06 mol, dichlorofluorescein per mol peroxynitrite at pH 8.5. The increase in yield of oxidized products as a function of pH indicates that the peroxynitrite anion may form an adduct with the indicator, followed by protonation and oxidation of the indicator. Carbon dioxide decreased the yield of fluorescent products to about 5%, relative to peroxynitrite, and the rate of product formation is again zero-order in the indicator. Given this yield, it is proposed that nitrogen dioxide and trioxocarbonate (*1-) are the reactive species that oxidize the indicators.     

Microsomal conjugation and oxidation of bilirubin

Bilirubin diglucuronide and bilirubin monoglucuronide are formed on incubation of microsomal preparations from rat liver with bilirubin and UDPglucuronate. Microsomal diglucuronide formation is a two-step reaction: first monoglucuronide is formed and this is subsequently converted to diglucuronide. Both steps require UDPglucuronate and have a similar pH optimum at pH 7.8. Albumin inhibits the conversion of monoto diglucuronide. Factors favouring diglucuronide formation are: (a) low bilirubin concentration; (b) relatively high UDPglucuronate concentration; (c) complete removal of UDPglucuronyltransferase latency. For the latter, trypsin-treatment appeared superior over digitonin or UDP-N-acetylglucosamine. Trypsin-treatment had to be done under strictly anaerobic conditions. If trypsin treatment was done under aerobic conditions, reactive molecules were formed which initiated the rapid oxidation of bilirubin and its glucuronides. Microsomal oxidation of bilirubin and glucuronides also occurred in untreated and digitonin-treated microsomes and was stimulated by NADPH and by the cytochrome P-450 inhibitor, metyrapone. This suggests that lipid peroxides act as initiators of bilirubin oxidation. Indirect evidence was found that trypsin inactivates nucleotide pyrophosphatase. This is an active UDPglucuronate-consuming enzyme in microsomal preparations which must be inactivated before meaningful kinetic studies can be done. With trypsin-treated microsomal preparations the Vmax for bilirubin monoglucuronide formation was 1.7 X 10(-9) mol . mg protein-1 . min-1 and KUDPglucuronatem 43 X 10(-6) M. For bilirubin diglucoronide formation the apparent Vmax was 0.7 X 10(-9) mol . mg protein-1 . min-1 and the apparent KUDPglucuronate m 1.0 X 10(-3) M.     

Spatial separation of anaerobic ammonium oxidation and nitrite-dependent anaerobic methane oxidation in permeable riverbeds

Anaerobic ammonium oxidation (anammox) and nitrite-dependent anaerobic methane oxidation (n-damo) play important roles in nitrogen and carbon cycling in fresh waters but we do not know how these two processes compete for their common electron acceptor, nitrite. Here, we investigated the spatial distribution of anammox and n-damo across a range of permeable riverbed sediments. Anammox activity and gene abundance were detected in both gravel and sandy riverbeds and showed a simple, common vertical distribution pattern, while the patterns in n-damo were more complex and n-damo activity was confined to the more reduced, sandy riverbeds. Anammox was most active in surficial sediment (0–2 cm), coincident with a peak in hzsA gene abundance and nitrite. In contrast, n-damo activity peaked deeper down (4–8 cm) in the sandy riverbeds, coincident with a peak in n-damo 16S rRNA gene abundance and higher methane concentration. Pore water nitrite, methane and oxygen were key factors influencing the distribution of these two processes in permeable riverbeds. Furthermore, both anammox- and n-damo- activity were positively correlated with denitrification activity, suggesting a role for denitrification in supplying both processes with nitrite. Our data reveal spatial separation between anammox and n-damo in permeable riverbed sediments that potentially avoids them competing for nitrite.     

Mild oxidation promotes and advanced oxidation impairs remodeling of human high-density lipoprotein in vitro

High-density lipoproteins (HDLs) prevent atherosclerosis by removing cholesterol from macrophages and by exerting antioxidant and anti-inflammatory effects. Oxidation is thought to impair HDL functions, yet certain oxidative modifications may be advantageous; thus, mild oxidation reportedly enhances cell cholesterol uptake by HDL whereas extensive oxidation impairs it. To elucidate the underlying energetic and structural basis, we analyzed the effects of copper and hypochlorite (which preferentially oxidize lipids and proteins, respectively) on thermal stability of plasma spherical HDL. Circular dichroism, light scattering, calorimetry, gel electrophoresis, and electron microscopy showed that mild oxidation destabilizes HDL and accelerates protein dissociation and lipoprotein fusion, while extensive oxidation inhibits these reactions; this inhibition correlates with massive protein cross-linking and with lipolysis. We propose that mild oxidation lowers kinetic barriers for HDL remodeling due to diminished apolipoprotein affinity for lipids resulting from oxidation of methionine and aromatic residues in apolipoproteins A-I and A-II followed by protein cross-linking into dimers and/or trimers. In contrast, advanced oxidation inhibits protein dissociation and HDL fusion due to lipid redistribution from core to surface upon lipolysis and to massive protein cross-linking. Our results help reconcile the apparent controversy in the studies of oxidized HDL and suggest that mild oxidation may benefit HDL functions.     

Myeloperoxidase oxidation states involved in myeloperoxidase-oxidase oxidation of thiols.

The human red-blood-cell glyoxalase system was modified by incubation with high concentrations of glucose in vitro. Red-blood-cell suspensions (50%, v/v) were incubated with 5 mM- and 25 mM-glucose to model normal and hyperglycaemic glucose metabolism. There was an increase in the flux of methylglyoxal metabolized to D-lactic acid via the glyoxalase pathway with high glucose concentration. The increase was approximately proportional to initial glucose concentration over the range studied (5-100 mM). The activities of glyoxalase I and glyoxalase II were not significantly changed, but the concentrations of the glyoxalase substrates, methylglyoxal and S-D-lactoylglutathione, and the percentage of glucotriose metabolized via the glyoxalase pathway, were significantly increased. The increase in the flux of intermediates metabolized via the glyoxalase pathway during periodic hyperglycaemia may be a biochemical factor involved in the development of chronic clinical complications associated with diabetes mellitus.