David Sheehan

Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily.

The glutathione transferases (GSTs; also known as glutathione S-transferases) are major phase II detoxification enzymes found mainly in the cytosol. In addition to their role in catalysing the conjugation of electrophilic substrates to glutathione (GSH), these enzymes also carry out a range of other functions. They have peroxidase and isomerase activities, they can inhibit the Jun N-terminal kinase (thus protecting cells against H(2)O(2)-induced cell death), and they are able to bind non-catalytically a wide range of endogenous and exogenous ligands. Cytosolic GSTs of mammals have been particularly well characterized, and were originally classified into Alpha, Mu, Pi and Theta classes on the basis of a combination of criteria such as substrate/inhibitor specificity, primary and tertiary structure similarities and immunological identity. Non-mammalian GSTs have been much less well characterized, but have provided a disproportionately large number of three-dimensional structures, thus extending our structure-function knowledge of the superfamily as a whole. Moreover, several novel classes identified in non-mammalian species have been subsequently identified in mammals, sometimes carrying out functions not previously associated with GSTs. These studies have revealed that the GSTs comprise a widespread and highly versatile superfamily which show similarities to non-GST stress-related proteins. Independent classification systems have arisen for groups of organisms such as plants and insects. This review surveys the classification of GSTs in non-mammalian sources, such as bacteria, fungi, plants, insects and helminths, and attempts to relate them to the more mainstream classification system for mammalian enzymes. The implications of this classification with regard to the evolution of GSTs are discussed.

Effects of seasonality on xenobiotic and antioxidant defence mechanisms of bivalve molluscs.

Levels of chemical pollutants in the environment often display wide seasonal variation in response to climatic and other factors. Use of bioindicators such as enzyme activities in biomonitoring studies is complicated by this variation. Many such enzyme activities themselves show considerable seasonal fluctuation and there is known to be seasonality also in natural exposure to oxidative stress. This review attempts to explore some consequences of seasonal variation for biomonitoring studies with bivalve molluscs. It is suggested that independence of seasonal variation should be seen as a desirable feature of a bioindicator molecule. Where such molecules show seasonal variation, however, this should be incorporated into interpretation of biomonitoring studies by the use of appropriate controls.

Oxidative stress and toxicity of gold nanoparticles in Mytilus edulis

Gold nanoparticles (AuNP) have potential applications in drug delivery, cancer diagnosis and therapy, food industry and environment remediation. However, little is known about their potential toxicity or fate in the environment. Mytilus edulis was exposed in tanks to 750 ppb AuNP (average diameter 5.3 ± 1 nm) for 24h to study in vivo biological effects of nanoparticles. Traditional biomarkers and an affinity procedure selective for thiol-containing proteins followed by two-dimensional electrophoresis (2DE) separations were used to study toxicity and oxidative stress responses. Results were compared to those obtained for treatment with cadmium chloride, a well known pro-oxidant. M. edulis mainly accumulated AuNP in digestive gland which also showed higher lipid peroxidation. One-dimensional SDS/PAGE (1DE) and 2DE analysis of digestive gland samples revealed decreased thiol-containing proteins for AuNP. Lysosomal membrane stability measured in haemolymph gave lower values for neutral red retention time (NRRT) in both treatments but was greater in AuNP. Oxidative stress occurred within 24h of AuNP exposure in M. edulis. Previously we showed that larger diameter AuNP caused modest effects, indicating that nanoparticle size is a key factor in biological responses to nanoparticles. This study suggests that M. edulis is a suitable model animal for environmental toxicology studies of nanoparticles.

Antioxidative effect of added tea catechins on susceptibility of cooked red meat, poultry and fish patties to lipid oxidation

The comparative antioxidant activity of added tea catechins on susceptibility of cooked and overwrapped red meat (beef and pork), poultry (chicken, duck and ostrich) and fish (whiting and mackerel) to lipid oxidation was investigated. Fresh meats, poultry and fish, purchased from a local market, were trimmed to remove bones, skin and visible fat and minced through a 4-mm plate. The minced muscle from each species was treated with either 1% NaCl (S), 300 mg tea catechins kg−1 minced muscle (TC) or 1% NaCl plus 300 mg tea catechins kg−1 minced muscle (TCS). Control minced muscle samples (C) contained neither NaCl nor tea catechins. Patties (50 g), prepared from treated and untreated minced muscle, were cooked until the core temperature reached 75°C, cooled down to room temperature and held in a refrigerated (4°C) and illuminated (616 lux) display cabinet for 10 days. Oxidative stability (TBARS) was measured at 3-day intervals. The susceptibility of cooked patties to lipid oxidation was closely related to lipid content, concentration of unsaturated fatty acids and presence of iron in different species. Addition of NaCl to raw minced muscle significantly (P<0.05) promoted lipid oxidation for cooked patties regardless of species sources. Tea catechins added at a level of 300 mg kg−1 minced muscle significantly (P<0.01) inhibited the pro-oxidation caused by NaCl and controlled lipid oxidation for all cooked muscle patties examined. Tea catechins at concentrations greater than 300 mg kg−1 were necessary to reduce oxidation for mackerel patties containing high levels of lipids and unsaturated fatty acids. The high affinity of tea catechins for the lipid bilayers of muscle and the radical scavenging abilities of tea catechins may be possible mechanisms to explain the oxidative stability in cooked muscle foods.

Seasonal variation in the antioxidant defence systems of gill and digestive gland of the blue mussel, Mytilus edulis

Abstract Seasonal variation in the level of glutathione (GSH) and in the activities of glutathione S-transferase (GST; EC 2.5.1.18), GSH-peroxidase (EC 1.11.1.9) and catalase (EC 1.11.1.6) in both the gill and digestive gland of the blue mussel (Mytilus edulis) sampled from Cork harbour were evaluated. GSH peroxidase activities are generally lowest in the period February–April. Catalase activity did not vary greatly over the year in the two tissues studied from two separate sampling sites. Levels are generally lower in the period December–April than in May–July. GSH levels fluctuated in both tissues with lowest levels occurring during the winter period. GST activity increases in the period September–March in gill tissue but does not vary significantly in digestive gland where levels are consistently lower. This enzyme may therefore carry out a tissue-specific role in protecting gill tissue against oxidative stress in winter.

Gold nanoparticles and oxidative stress in Mytilus edulis

Little is known about potential environmental impact of nanoparticles. Gold nanoparticles can cause unexpected biological responses. Here, Mytilus edulis were exposed (24h) to gold-citrate nanoparticles (GNP), menadione and both compounds simultaneously (GNP/menadione). Protein ubiquitination and carbonylation were determined in gill, mantle and digestive gland, along with traditional oxidative stress biomarkers; catalase activity and neutral red retention time assay (haemolymph). 2DE gels were performed on gill proteins (menadione; GNP/menadione). Our results reveal that GNP may induce oxidative stress.

Dietary tea catechins and iron-induced lipid oxidation in chicken meat, liver and heart

The effects of dietary tea catechins (TC) supplementation at levels of 50 (TC 50), 100 (TC 100), 200 (TC 200), and 300 (TC 300) mg kg(-1) feed on susceptibility of chicken breast meat, thigh meat, liver and heart to iron-induced lipid oxidation were investigated. Day old chicks (n=200) were randomly divided into six groups. Chicks were fed diets containing either basal (C), or α-tocopheryl acetate supplementation at a level of 200 mg kg(-1) feed (VE 200), or TC supplementation for 6 weeks prior to slaughter. Lipid oxidation was assessed by monitoring malondialdehyde formation with 2-thiobarbituric acid (TBA) assay. TC supplementation at all levels exerted antioxidative effects for all tissues with the exception of 50 mg kg(-1) feed for breast meat. TC supplementation at levels of 200 and 300 mg kg(-1) feed were found to be significantly (P<0.05) more effective in retarding lipid oxidation in all tissues, compared to the control. TC supplementation at a level of 300 mg kg(-1) feed was also found to be significantly (P<0.05) superior to vitamin E supplementation at a level of 200 mg kg(-1) feed (VE 200) for oxidative stability in chicken thigh meat, but it was inferior to VE 200 in chicken liver and heart. TC supplementation at a level of 50 mg kg(-1) feed was found to be pro-oxidative in breast meat, but this did not occur in chicken thigh meat, liver and heart. The variation of TC antioxidative properties in different tissues may be explained by the uneven distribution of lipid, iron and TC accumulation in tissues.

Antioxidative effect of dietary tea catechins on lipid oxidation of long-term frozen stored chicken meat

The antioxidative effect of dietary tea catechins (TC) supplementation at levels of 50, 100, 200 and 300 mg kg(-1) feed on susceptibility of chicken breast and thigh meat to lipid oxidation during frozen (-20°C) storage for 9 months was investigated. Day-old chickens (Cobb 500, n=200) were randomly divided into six groups. Chickens were fed a basal diet containing 20 mg α-tocopheryl acetate kg(-1) feed as control, or a vitamin E supplemented diet (basal diet plus 200 mg α-tocopheryl acetate kg(-1) feed), or TC supplemented diets (basal diet plus 50, 100, 200 or 300 mg TC kg(-1) feed) for 6 weeks prior to slaughter. Lipid oxidation (TBARS) was assessed after 0 and 10 days of refrigerated display (4°C) following 1, 3, 6, and 9 months of frozen (-20°C) storage. TC supplementation at all concentrations showed antioxidative effects for both breast and thigh chicken meat during the 9 months of frozen storage compared to the control sample. TC supplementation at levels of 200 and 300 mg kg(-1) feed were more effective (P<0.05) in delaying lipid oxidation in all meat samples compared to the control. TC supplementation at a level of 200 mg kg(-1) feed showed antioxidant activity equivalent to α-tocopheryl acetate fed at the same level up to 3 months of frozen storage. For long-term frozen storage up to 9 months, however, TC supplementation at 300 mg kg(-1) feed was required as a replacement for α-tocopheryl acetate at a level of 200 mg kg(-1) feed. The results obtained showed a long-term antioxidative effect exhibited by dietary tea catechins on chicken meat during frozen storage and demonstrated that tea catechins are effective alternatives to vitamin E as natural dietary antioxidants.

Separation of multiple forms of glutathione S-transferase from the Blue Mussel, Mytilus edulis

1. Glutathione S-transferase isoenzymes from Mytilus edulis and M. galloprovincialis have been partially purified by glutathione-sepharose affinity chromatography followed by Mono Q anion exchange fast protein liquid chromatography (f.p.l.c.). 2. The tissue distribution of glutathione S-transferase in M. edulis has been studied. Using 1-chloro-2,4-dinitrobenzene as substrate, highest specific activity is observed in the gill, the main feeding organ. Affinity-purified extracts of this organ give a characteristic f.p.l.c. profile. A similar profile is obtained with affinity-purified extracts of the digestive gland of M. galloprovincialis. 3. The subunit structure of the purified isoenzymes has been studied by SDS polyacrylamide gel electrophoresis and reversed-phase h.p.l.c. The subunits have similar molecular weights and h.p.l.c. retention times to rat glutathione S-transferases.

Exposure of the blue mussel, Mytilus edulis, to gold nanoparticles and the pro-oxidant menadione

Relatively little is known about how gold nanoparticles (GNP) might interact in vivo with marine organisms. Mytilus edulis was exposed (24h) to approximately 15 nm GNP, menadione and both compounds simultaneously (GNP/menadione). GNP was detected by inductively coupled plasma-optical emission spectroscopy mainly in digestive gland of samples exposed to GNP though not GNP/menadione, perhaps due to impaired feeding. Thioredoxin reductase activity and malondialdehyde levels were determined in all tissues. Thioredoxin reductase inhibition was detected only in digestive gland exposed to menadione whilst malondialdehyde levels did not vary in response to treatment in all tissues. GNP caused a decrease in the reduced/oxidized glutathione ratio in digestive gland, but no difference was found in other tissues or for other treatments. One dimensional electrophoresis of proteins containing thiol groups was performed in all tissues and revealed a reduction in protein thiols for all treatments in digestive gland. Two dimensional electrophoresis of digestive gland extracts, from GNP and control groups, showed decreased levels of thiol proteins in response to GNP which we attribute to oxidation. Our results suggest that GNP causes a modest level of oxidative stress sufficient to oxidize thiols in glutathione and proteins but without causing lipid peroxidation or induction of thioredoxin reductase activity.