Gary W. Winston

Total oxidant scavenging capacity (TOSC) of microsomal and cytosolic fractions from Antarctic, Arctic and Mediterranean scallops: differentiation between three potent oxidants

The enhanced formation of reactive oxygen species (ROS) is a common pathway of toxicity induced by stressful environmental conditions. In polar environments, characterization of antioxidant defences in key sentinel species may be of particular value as early detection biomarkers of unforeseen effects of human activities which are progressively increasing in these remote areas.The complexities associated with predicting the consequences at the organism level of variations of specific antioxidant defences have been recently overcome by the ability to quantify an index of specific biological resistance to various kinds of ROS.The total oxyradical scavenging capacity (TOSC) assay has been used in three species of scallops for quantifying their ability to neutralize peroxyl (ROO(&z.rad;)) and hydroxyl (&z.rad;OH) radicals and peroxynitrite (HOONO). Adamussium colbecki and Chlamys islandicus represent key organisms for monitoring Antarctic and Arctic regions while Pecten jacobaeus was chosen for a comparison with a related temperate species. TOSC values for ROO&z.rad; were significantly higher in A. colbecki indicating this species as the most efficient scavenger of ROO&z.rad;. Mediterranean scallops had the lowest TOSC for ROO(&z.rad;). A. colbecki also exhibited the highest scavenging capacity for &z.rad;OH with values more than 2-fold greater than for C. islandicus and P. jacobaeus. TOSC for HOONO was lower for all scallops as compared to those for ROO&z.rad; or &z.rad;OH. TOSC for microsomes was not significantly different among the species for any ROS studied, and the percentage contribution to the specific TOSC for the various oxidants of microsomes of all scallops accounted for 1-3% of the total TOSC of the post-mitochondrial fraction. The specific TOSC of scallop microsomes for &z.rad;OH was approximately ten times lower than that for ROO&z.rad; or HOONO.The higher basal capability of the Antarctic scallop to neutralize different reactive oxygen species is discussed in terms of a possible adaptation to this extreme environment and TOSC is validated as a quantifiable measure of susceptibility to oxidative stress in marine organisms.

Seasonal variations of susceptibility to oxidative stress in Adamussium colbecki, a key bioindicator species for the Antarctic marine environment.

The area of free radical biology is of increasing interest for marine organisms since the enhanced formation of reactive oxygen species (ROS) is a common pathway of toxicity induced by stressful environmental conditions. In polar environments responses of the antioxidant system could be useful as an early detection biomarkers of unforeseen effects of human activities which are progressively increasing in these remote areas. However, the characterization of antioxidant defences in appropriate sentinel species is of particular value also in terms of a possible adaptation to this extreme environment. The scallop, Adamussium colbecki, is a key species for monitoring the Antarctic environment and, besides single antioxidants, the total oxyradical scavenging capacity (TOSC) assay has been recently used for quantifying the overall ability of this organism to neutralize peroxyl radicals (ROO*), hydroxyl radicals (*OH) and peroxynitrite (HOONO). The aim of this work was to obtain a better characterization of these biological responses which can indicate the occurrence of biological disturbance; in this study the total oxyradical scavenging capacity was further analyzed to assess the presence of seasonal fluctuations in the susceptibility to oxidative stress in this species. The capability to neutralize peroxyl radicals and hydroxyl radicals increased at the end of December, while resistance towards peroxynitrite did not show any significant variations during the Antarctic summer. These results suggest the occurrence of metabolic changes which mainly influence intracellular formation of ROO* and *OH, with more limited effects on HOONO. Despite the limited time window analyzed, as a typical constraint in Antarctic research at Terra Nova Bay, an increased resistance to these specific oxyradicals might be related to the period of highest feeding activity, or to other intrinsic factors in the animals physiology such as the phase of reproductive cycle.

Applications of a new method for measuring the total oxyradical scavenging capacity in marine invertebrates

Abstract A new method is described for measuring the total oxyradical scavenging capacity (TOSC) in biological tissues. The TOSC-assay is based on the reaction between peroxyl radicals and α-keto-γ-methiolbutyric acid which is oxidized to ethylene. The presence within the tissues of molecules that scavenge for peroxyl radicals generated by thermal homolysis of 2,2′ azo-bis-amidinopropane quantitatively reduce ethylene production. Cytosol from invertebrates showed a higher ROO ° absorbance capacity than that of rat liver and in all the species endogenous soluble antioxidants provided greater protection than proteins. The expected contribution of GSH, ascorbic acid and uric acid accounted for only 35% of the measured TOSC values. This method is useful for assessing the biological resistance to oxidative stress and its relationship to environmental pollutants in quantifiable terms.

Sex-related responses to oxidative stress in primary cultured hepatocytes of European flounder (Platichthys flesus L.)

Effects of oxidative stress induced by xenobiotic compounds were studied in primary cultures of isolated hepatocytes of immature European flounder (Platichthys flesus L.) of both sexes caught in a relatively unpolluted area of the German Bight (North Sea). Cells were exposed to oxidative stressors such as 100 microM hydrogen peroxide (H2O2), 100 microM benzo[a]pyrene (B[a]p) and 50 microM nitrofurantoin (N-(5-nitro-2-furfurylidene)-1-aminohydantoin; NF) for 2 and 24 h. Cell mortality was determined with the use of the fluorescent ethidium homodimer-1 and calcein. Oxidative stress response was assessed by quantitative analysis of (1) intracellular reactive oxygen species (ROS) formation with dihydrorhodamine 123, (2) lipid peroxidation on the basis of concentrations of lipid hydroperoxides and the lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) and (3) cellular total oxidant-scavenging capacity (TOSC) using the TOSC assay (Winston et al., 1998). An increase in ROS formation was detected as early as 2 h after exposure to H2O2, B[a]p and NF. After 24 h, stress responses were lower, except following exposure to NF. The pattern of responses differed with the different oxidative stressors. Lipid peroxidation and the capacity to scavenge ROS were increased significantly in both sexes only after exposure to H2O2, whereas B[a]p and NF provoked sex-dependent responses. B[a]p-induced lipid peroxidation and increase in scavenging capacity were observed only in hepatocytes of females, whereas NF initiated these responses only in cells of males. Sex differences in oxidative stress response only after exposure to pro-oxidants that require enzymatic activation infer the importance of biotransformation pathways in stress responses. Because of their sensitivity to oxidative stress, flounder hepatocytes provide a useful model for early risk assessment of xenobiotics.

Total oxyradical scavenging capacity in mussel Mytilus sp. As a new index of biological resistance to oxidative stress

The Total Oxyradical Scavenging Capacity (TOSC) has been characterized in various subcellular fractions from digestive gland of the mussel Mytilus sp.. A simple and sensitive gas-chromatographic assay is used for quantitating this new biochemical parameter. n nThe method is based on the reaction between peroxyl radicals and α-keto-γ-methiolbutyric acid (KMBA) which is oxidized to ethylene: in the presence of cellular antioxidant competing with KMBA for peroxyl radicals, ethylene formation is quantitatively reduced. n nWhole cytosol from M. edulis exhibited slightly higher TOSC values than the Mediterranean species. Soluble antioxidants provided greater protection than proteins accounting for 70% of oxyradical scavenging capacity of whole cytosols. TOSC assay was suitable also for lipid-soluble antioxidants, and the analysis of the microsomal fraction confirmed the importance of membrane-associated antioxidants in protection against toxicity of reactive oxygen species (ROS). n nThe analysis of TOSC in sentinel organisms is proposed as a new index of contaminant exposure, one which permits quantifiable assessment of the biological resistance to oxidative stress.

Aldehyde dismutase activity of yeast alcohol dehydrogenase

Yeast alcohol dehydrogenase (ECxa01.1.1.1) is able to catalyze the oxidation of acetaldehyde by NAD+ with a concomitant formation of ethanol, at pHxa08.8 and pHxa07.1; the stoichiometry of aldehyde oxidation vs. ethanol formation is 2:1. This enzymatic reaction obeys the Michaelis-Menten kinetics and was characterized by a high KM for acetaldehyde (68xa0mM) and a low kcat (2.3xa0s−1), at pHxa08.8, 22u2009°C. There is no visible burst of NADH during the reaction, from pHxa07.1–10.1. Therefore, we have concluded that the enzyme catalyzes an apparent dismutation of two molecules of acetaldehyde into a molecule of acetic acid and a molecule of ethanol.

Distribution of microsomal CO-binding chromophores and EROD activity in sea anemone tissues

Our previous studies indicated that sea anemone microsomes contain cytochrome P450 (CYP) and have ethoxyresorufin O-dealkylation (EROD) activity. Other marine invertebrates have discrete organs which concentrate cytochromes P450, whereas cnidarians have evolved only to the tissue level of development. To examine the distribution of CYP in sea anemones, microsomes were prepared from the following tissue regions of two sea anemones, Anthopleura xanthogrammica: outer (heavy muscular wall), inner (imperfect and perfect mesentery, and retractor muscle), soft (digestive sac, gonads, and mesentery filaments), and tentacular (including algal/diatom symbiont). The cytochrome P450 content was distributed relatively evenly among the tissue regions. In contrast, the 418-nm CO-binding chromophore was approximately 10 times greater in the outer region than in any other region. Further, the 490-nm peak (which interferes with quantification of CYP in sea anemones) was greater in the outer region. In general, the EROD activity was comparable in the inner and soft regions and highest in the tentacles. However, the EROD results may have been complicated by the presence of the algal/diatom symbiont.

Oxidation of thiodiglycol (2,2'-thiobis-ethanol) by alcohol dehydrogenase: comparison of human isoenzymes.

Sulfur mustard is a chemical warfare agent that causes blistering of the skin and damages the eyes and airway after environmental exposure. We have previously reported that thiodiglycol (TDG, 2,2′‐bis‐thiodiethanol), the hydrolysis product of sulfur mustard, is oxidized by alcohol dehydrogenase (ADH) purified from horse liver or present in mouse liver and human skin cytosol. Humans express four functional classes of ADH composed of several different isozymes, which vary in their tissue distribution, some occurring in skin. To help us evaluate the potential contribution of the various human isozymes toward toxicity in skin and in other tissues, we have compared the catalytic activity of purified human class I αα‐, β1β1‐, β2β2‐, and γ1γ1‐ADH, class II π‐ADH, class III χ‐ADH, and class IV σ‐ADH with respect to TDG oxidation and their relative sensitivities to inhibition by pyrazole. Specific activities toward TDG were 123, 79, 347, 647, and 12 nmol/min/mg for the class I αα‐, β1β1‐, β2β2‐, and γ1γ1‐ADH and class II π‐ADH, respectively. TDG was not a substrate for class III χ‐ADH. The specific activity of class IV σ‐ADH was estimated at about 1630 nmol/min/mg. 1 mM pyrazole, a potent inhibitor of class I ADH, inhibited the class I αα, β1β1, β2β2, and γ1γ1 ADH and class IV σ‐ADH by 83, 100, 56, 90, and 73%, respectively. The class I αα‐ and β1β1‐ADH oxidized TDG with kcat/Km value of 7–8 mM−1 min−1, β2β2‐ADH with a value 19 mM−1 min−1 and class I γ1γ1‐ADH with a value of 176 mM−1 min−1. The kcat/Km value for class IV σ‐ADH was estimated at 4 mM−1 min−1. The activities of class IV σ‐ADH and class I γ1γ1‐ADH are of significant interest because of their prevalence in eyes, lungs, stomach, and skin, all target organs of sulfur mustard toxicity.

Total oxidant scavenging capacity of Antarctic, Arctic, and Mediterranean scallops

Abstract The Total Oxyradical Scavenging Capacity (TOSC) assay was used in three species of scallops for quantifying their ability to neutralise peroxyl (ROO*) and hydroxyl (*OH) radicals and per‐oxynitrite (HOONO). Adamussium colbecki and Chlamys islandicus represent key organisms for monitoring Antarctic and Arctic regions while Pecten jacobaeus was chosen for comparison with a related temperate species. TOSC values for ROO* were significantly higher in A. colbecki, indicating that this species is the most efficient ROO* scavenger. Mediterranean scallops had the lowest TOSC for ROO*. Adamussium colbecki also exhibited the highest scavenging capacity for *OH with values more than two‐fold greater than for C. islandicus and P. jacobaeus. TOSC values for HOONO were lower for all scallops as compared to those for ROO* or *OH; for microsomes they were not significantly different among the species for any reactive oxygen species studied, and the percentage contribution to the specific TOSC for the various oxidants of microsomes of all scallops accounted for 1–3% of the total TOSC of the post‐mitochondrial fraction. The specific TOSC of scallop microsomes for *OH was ‐10 times lower than that for ROO* or HOONO. The higher basal capability of the Antarctic scallop to neutralise different reactive oxygen species is discussed in terms of a possible adaptation to this extreme environment, and TOSC is validated as a quantifiable measure of susceptibility to oxidative stress in marine organisms. In a second part of this study, the seasonal fluctuations of susceptibility to oxidative stress were analysed in the Antarctic scallop Adamussium colbecki by the TOSC Assay. A marked increase in the capability to neutralise peroxyl radicals and hydroxyl radicals was demonstrated at the end of December, while resistance towards peroxynitrite did not show significant variations during the Antarctic summer. The increased resistance to these specific oxyradicals was almost contemporary with the period of highest feeding activity and the development of gonadic tissues. Sensitivity to hydroxyl radicals and peroxyl radicals toxicity was also influenced by UV‐B and UV‐? wavelengths, as indicated by the lower TOSC values in scallops exposed under laboratory conditions. Toxicity of UV radiation seems partly mediated by formation of reactive oxygen species caused by the direct exposure of mantle edges and/or the photochemical production of hydrogen peroxide in sea water.

A novel substrate for yeast alcohol dehydrogenase – p-nitroso-N,N-dimethylaniline

Yeast alcohol dehydrogenase (EC 1.1.1.1) catalyzes the novel reduction of p-nitro-so-N,N-dimethylaniline with NADH as a cofactor. Apparent kinetic constants for this enzymatic reaction are: V2=2.1 s−1, KQ=456 μM, KiQ=119 μM, and KP=1.47 mM, at pH 8.9, 25 °C. This reaction is especially useful for the quantitative determination of NAD+ and NADH by enzymatic cycling.