Steven P. Gieseg

Reactive species and their accumulation on radical-damaged proteins

Hydroperoxides and catechols are described as novel reactive products of radical attack on proteins. These species, like other components of oxidized and otherwise damaged proteins, may accumulate in some biological systems. We propose that the reactive species may then attack other biomolecules, and constitute both a marker and a mechanism of age-related pathologies.

Low density lipoprotein is saturable by pro-oxidant copper

The oxidative resistance of low density lipoprotein (LDL) can be experimentally described by the length of time during which no significant lipid peroxidation is observed in a pro‐oxidant environment. This period of inhibited oxidation, termed the ‘lag phase’, is partially due to the radical scavenging reactions of the anti‐oxidants contained in the LDL particle. We have shown that the LDL lag time decreases with increasing copper concentration, leveling out at a relatively high copper‐to‐LDL ratio. This behaviour demonstrates the existence of a finite number of saturable pro‐oxidant copper binding sites within the LDL particle. The relationship is described by the equation, lag time = [Cu]− · K · t min + t min where the constant, K, is the negative reciprocal of the x‐axis intercept of the graphed function, and t min is given by the y‐axis intercept. By this definition of the constant, K is the amount of copper that will produce a lag time of twice t min, while t min is the minimum time a particular LDL will resist oxidation at a maximum copper concentration.

The participation of nitric oxide in cell free- and its restriction of macrophage-mediated oxidation of low-density lipoprotein

The potential role of nitric oxide radical (NO .) in macrophage-mediated oxidation and conversion of human low density lipoprotein (LDL) to a high-uptake form was examined by exposing LDL to aerobic solutions of either NO . or 3-morpholino-sydnonimine-hydrochloride (SIN-1, a compound that spontaneously forms NO . and superoxide anion radical) or to mouse peritoneal macrophages in the presence and absence of modulators of cellular NO . synthesis. Incubation with NO . alone caused oxidation of LDLs ubiquinol-10 and accumulation of small amounts of lipid hydroperoxides, but failed to form any high-uptake ligand for endocytosis by macrophages and did not alter the LDL particle charge or the integrity of apoB. Exposure of LDL to SIN-1 resulted in complete consumption of all antioxidants and substantial formation of lipid hydroperoxides, but again had little effect on the lipoprotein particle charge or generation of high-uptake form. Preincubation of macrophages with interferon-gamma increased the cells ability to generate reactive nitrogen metabolites. The extent of cell-mediated oxidation of LDL and the generation of high-uptake LDL was substantial in resident cells in which NO . synthesis was barely detectable, depressed in cells active in NO . synthesis and restored when NO . synthesis was suppressed by the arginine analogue, NMMA. These results suggest that, while together with superoxide anion radical, NO . can oxidize LDL, its synthesis is not required for macrophage-mediated oxidation of LDL in vitro; rather it exerts a protective role in preventing oxidative LDL modification by macrophages.

Peroxidation of proteins before lipids in U937 cells exposed to peroxyl radicals

This study provides the first report of the formation of protein hydroperoxides in cells attacked by reactive oxygen species. U937 cells exposed to peroxyl radicals generated by the thermal decomposition of a water-soluble azo compound gradually accumulated hydroperoxide (-OOH) groups. In an incubation for 22 h, 1.2 mM peroxyl radicals was generated and each cell acquired 1.5x10(8) -OOH groups. These groups were located on the cell proteins; no lipid peroxidation was detected. The extent of protein peroxidation was proportional to the rate of generation of the peroxyl radicals. There was no lag period before the onset of peroxidation, indicating that cell antioxidants could not protect the proteins. The half-life of protein hydroperoxides in cell suspensions was approx. 4 h at 37 degrees C. Our results suggest that protein hydroperoxides might have a significant role as intermediates in the development of biological damage initiated by reactive oxygen species.

Warming, CO2, and nitrogen deposition interactively affect a plant‐pollinator mutualism

Environmental changes threaten plant-pollinator mutualisms and their critical ecosystem service. Drivers such as land use, invasions and climate change can affect pollinator diversity or species encounter rates. However, nitrogen deposition, climate warming and CO(2) enrichment could interact to disrupt this crucial mutualism by altering plant chemistry in ways that alter floral attractiveness or even nutritional rewards for pollinators. Using a pumpkin model system, we show that these drivers non-additively affect flower morphology, phenology, flower sex ratios and nectar chemistry (sugar and amino acids), thereby altering the attractiveness of nectar to bumble bee pollinators and reducing worker longevity. Alarmingly, bees were attracted to, and consumed more, nectar from a treatment that reduced their survival by 22%. Thus, three of the five major drivers of global environmental change have previously unknown interactive effects on plant-pollinator mutualisms that could not be predicted from studies of individual drivers in isolation.

Protein hydroperoxides are a major product of low density lipoprotein oxidation during copper, peroxyl radical and macrophage-mediated oxidation.

Damage to apoB100 on low density lipoprotein (LDL) has usually been described in terms of lipid aldehyde derivatisation or fragmentation. Using a modified FOX assay, protein hydroperoxides were found to form at relatively high concentrations on apoB100 during copper, 2,2′-azobis(amidinopropane) dihydrochloride (AAPH) generated peroxyl radical and cell-mediated LDL oxidation. Protein hydroperoxide formation was tightly coupled to lipid oxidation during both copper and AAPH-mediated oxidation. The protein hydroperoxide formation was inhibited by lipid soluble α-tocopherol and the water soluble antioxidant, 7,8-dihydroneopterin. Kinetic analysis of the inhibition strongly suggests protein hydroperoxides are formed by a lipid-derived radical generated in the lipid phase of the LDL particle during both copper and AAPH mediated oxidation. Macrophage-like THP-1 cells were found to generate significant protein hydroperoxides during cell-mediated LDL oxidation, suggesting protein hydroperoxides may form in vivo within atherosclerotic plaques. In contrast to protein hydroperoxide formation, the oxidation of tyrosine to protein bound 3,4-dihydroxyphenylalanine (PB-DOPA) or dityrosine was found to be a relatively minor reaction. Dityrosine formation was only observed on LDL in the presence of both copper and hydrogen peroxide. The PB-DOPA formation appeared to be independent of lipid peroxidation during copper oxidation but tightly associated during AAPH-mediated LDL oxidation.

Potential to inhibit growth of atherosclerotic plaque development through modulation of macrophage neopterin/7,8-dihydroneopterin synthesis.

The rise in plasma neopterin observed with increasing severity of vascular disease is a strong indicator of the inflammatory nature of atherosclerosis. Plasma neopterin originates as the oxidation product of 7,8‐dihydroneopterin secreted by γ‐interferon stimulated macrophages within atherosclerotic plaques. Neopterin is increasingly being used as a marker of inflammation during clinical management of patients with a range of disorders including atherosclerosis. Yet the role of 7,8‐dihydroneopterin/neopterin synthesis during the inflammatory process and plaque formation remains poorly understood and controversial. This is partially due to the unresolved role oxidants play in atherosclerosis and the opposing roles of 7,8‐dihydroneopterin/neopterin. Neopterin can act as pro‐oxidant, enhancing oxidant damage and triggering apoptosis in a number of different cell types. Neopterin appears to have some cellular signalling properties as well as being able to chelate and enhance the reactivity of transition metal ions during Fenton reactions. In contrast, 7,8‐dihydroneopterin is also a radical scavenger, reacting with and neutralizing a range of reactive oxygen species including hypochlorite, nitric oxide and peroxyl radicals, thus protecting lipoproteins and various cell types including macrophages. This has led to the suggestion that 7,8‐dihydroneopterin is synthesized to protect macrophages from the oxidants released during inflammation. The oxidant/antioxidant activity observed in vitro appears to be determined both by the relative concentration of these compounds and the specific chemistry of the in vitro system under study. How these activities might influence or modulate the development of atherosclerotic plaque in vivo will be explored in this review.

A comparison of plasma vitamin C and E levels in two Antarctic and two temperate water fish species

Antarctic fish have a high polyunsaturated lipid content and their muscle cells have a high mitochondria density suggesting that Antarctic fish are under greater oxidative stress than temperate water fish. To test this hypothesis, the plasma concentrations of the antioxidant vitamins E and C were measured in two Antarctic fish species, Pagothenia borchgrevinki and Trematomus bernacchii, and compared with the plasma concentrations of these vitamins in two New Zealand temperate water fish species, blue cod (Parapercis colias) and banded wrasse (Notolabrus fucicola). Neither vitamin is known to be synthesised in fish and so must be obtained from the diet. The plasma from both Antarctic fish species had vitamin E concentrations five to six times higher than those found in the two temperate water fish species. However, significantly higher levels of vitamin C were only found in the plasma of T. bernacchii, a benthic Antarctic fish. The average level of vitamin C in the plasma of the cryopelagic P. borchgrevinki was approximately one-third that of T. bernacchii. The T. bernacchii plasma yielded a high range of vitamin C values, possibly reflecting differences in nutritional status among the animals captured. No beta-carotene was found in any of the fish plasma samples studied. The data suggest that even though Antarctic fish live at -1.5 degrees C they may be exposed to greater metabolic stress from free radical mediated oxidation than temperate water species.

Factors affecting resistance of low density lipoproteins to oxidation

Oxidation resistance (OR) of low density lipoproteins (LDL) is frequently determined by the conjugated diene (CD) assay, in which isolated LDL is exposed to Cu2+ as prooxidant in the range of 1–10 μM. A brief review on major findings obtained with this assay will be given. A consistent observation is that vitamin E supplements or oleic acid-rich diets increase OR. Oxidation indices measured by the CD assay and effects of antioxidants very significantly depend on the Cu2+ concentration used for LDL oxidation. For medium and high Cu2+ concentrations, the relationship between lag time and propagation rate can be described by a simple hyperbolic saturation function, which has the same mathematical form as the Michaelis-Menten equation. At medium and high Cu2+ concentrations (0.5 to 5 μM), vitamin E increases lag time in a dose-dependent manner. The increase is higher for 0.5 μM Cu2+ as compared to 5 μM. At low Cu2+ concentrations (0.5 μM or less), the mechanism of LDL oxidation changes. Significant oxidation occurs in a preoxidation phase, which commences shortly after addition of Cu2+. Preoxidation is not inhibited by vitamin E. It is concluded that much additional work is needed to validate the importance of oxidation indices derived from CD and similar assays.

Hypothesis: A damaging role in aging for reactive protein oxidation products?

This paper discusses our knowledge of protein oxidation and its relationship to aging. It also outlines new observations from our laboratories concerning reactive species produced during protein oxidation, and proposes that these may inflict damage on other molecules, and hence contribute to the progression of aging. Whereas it has previously been difficult to see how relatively inert protein oxidation products could possibly have any causal role in aging, the detection of these novel reactive species implies a potentially significant role.