Organofluorophosphate-hydrolyzing activity in an estuarine clam, Rangia cuneata

1. The bivalve Rangia cuneata can enzymatically detoxify the organophosphorus acetylcholinesterase inhibitors DFP and soman. 2. Digestive gland homogenates contained Mazur-type DFPases based on response to Mn2+ ions, and relative rates of DFP: soman hydrolysis. Squid-type DFPase contributed little to the total organophosphate acid (OPA) anhydrase activity of these preparations. 3. The natural substrate(s) and physiological role(s) of OPA anhydrase in R. cuneata has yet to be determined; however, DFPase specific activity was pronounced in the digestive gland, the primary organ involved in bioconcentration and biotransformation of xenobiotics, and in the gills, which are in continuous contact with water-borne chemicals.     

The role of phospholipase A2 in microsomal lipid peroxidation induced with t-butyl hydroperoxide

The role of phospholipase A2 (PlA2) in lipid peroxidation induced with t-butyl hydroperoxide was examined in rat liver microsomes. Exposure of microsomes to t-butyl hydroperoxide was associated with activation of endogenous PlA2. When PlA2 was inhibited with chlorpromazine, mepacrine, or p-bromphenacyl bromide, the accumulation of thiobarbituric acid reactive substances (TBARS) was reduced in a dose dependent manner. In contrast, the accumulation of conjugated dienes was not affected by chlorpromazine, and was slightly increased by mepacrine. When endogenous PlA2 was activated with mellitin prior to induction of peroxidation, accumulation of both TBARS and dienes was reduced. Analogously, pretreatment with exogenous PlA2 reduced both dienes and TBARS. In contrast, addition of mellitin following the induction of peroxidation did not alter either TBARS or dienes.     

Protective effect of ellagic acid on t-butyl hydroperoxide induced lipid peroxidation in isolated rat hepatocytes

Ellagic acid, a plant polyphenol, showed protective effect on isolated rat hepatocytes against destruction due to lipid peroxide formation induced by t-butyl hydroperoxide in vitro. Ellagic acid inhibited the generation of superoxide anions and hydroxyl radicals both in enzymic and non enzymic systems, thus providing protection against oxidative damage.     

Identification and comparison of the organophosphate acid anhydrase activities of the clam,Rangia cuneata

1. Tissue extracts of the commonly found brackish water clam Rangia cuneata were found to degrade the potent neurotoxin diisopropylfluorophosphate (DFP) and surprisingly N, N′-diisopropylphosphorodiamidofluoridate (mipafox).2. Results indicate two groups of molecular weight-estimates for substrate specific enzymes within the digestive gland of R. cuneata. When DFP was a substrate, a protein in the range of 30,500–21,300 D was identified as OPA anhydrase. With mipafox as substrate, an OPA anhydrase ranging in weight from 105,000 to 138,300 D was identified.3. This data suggests at least two forms of active OPA anhydrase type proteins are active within R. cuneata. Suggestions as to the natural role of the OPA anhydrases and the implications in predicting environmental toxicity and in hazardous waste site clean up are discussed.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals.     

Cytochrome P-450 deficiency and resistance to t-butyl hydroperoxide of hepatoma microsomal lipid peroxidation

Lipid peroxidation of microsomes from rat liver and Morris hepatoma 9618A was induced by means of tert-butyl hydroperoxide (t-BuOOH). In rat liver microsomes t-BuOOH stimulated an early formation of lipid hydroperoxides (LOOH) and an increasing accumulation of malondialdehyde; t-BuOOH was completely consumed and cytochrome P-450 was rapidly destroyed. In hepatoma microsomes (60% deficiency of cytochrome P-450) a remarkable inhibition of both malondialdehyde and LOOH was observed; t-BuOOH was consumed only partially and cytochrome P-450 was destroyed slowly. In the presence of aminopyrine, malondialdehyde production was inhibited to the same extent (about 70%) in normal and tumour microsomes. The concentration of t-BuOOH required to achieve half-maximal velocity of malondialdehyde accumulation was comparable in the two microsome types. It is proposed that the deficiency of cytochrome P-450 limits the activation of t-BuOOH to the free radical species which initiate lipid peroxidation. Low cytochrome P-450 content would also affect the LOOH-dependent propagation of lipid peroxidation.     

Aging and lung antioxidant enzymes, glutathione, and lipid peroxidation in the rat.

The five major antioxidants enzymes, cytochrome oxidase (COX), GSH, and GSSG, and endogenous and in vitro stimulated lipid peroxidation (TBA-RS) were assayed in the lung of old (28 months) and young (9 months) adult rats due to the almost total absence of data of this kind in this tissue, which is normally exposed to relatively high pO2 throughout life. Catalase, selenium (Se)-dependent GSH peroxidase (GPx), GSH reductase, GSH, GSSG, GSSG/GSH, and in vivo and in vitro TBA-RS showed similar values in old and young animals. The decrease observed for non Se-dependent GPx disappeared when the values were expressed in relation to COX activity. Only superoxide dismutase showed a clear decrease when referred both to protein and COX activity. These results suggest that lung aging is not accelerated in old age due to a decrease in the antioxidant capacity of the tissue. Nevertheless, they are compatible with a continuous damage of the lung tissue by free radicals throughout the life span.     

Effect of multiple-locus heterozybosity and salinity on clearance rate in a brackish-water clam,Rangia cuneata (Sowerby)

Previous studies of several species of marine bivalves and gastropods have reported a positive correlation between growth or size and level of multiple-locus heterozygosity. There is some evidence that the growth advantage of relatively heterozygous individuals is due to a lower rate of standard or routine metabolism, compared with more homozygous individuals, although heterozygosity-dependent differences in feeding rate may also be involved. The present study examined the relationship between clearance rate in three salinity treatments (5,15, and 25%.) and multiple-locus heterozygosity at nine polymorphic allozyme loci in the clam Rangia cuneata (Sowerby). Clearance rates were determined by disappearance of an algal suspension from a flowing-water system. Allozyme genotypes were determined using starch-gel electrophoresis. The polymorphic loci examined were those coding for a nonspecific esterase (Est), mannosephosphate isomerase (Mpi), leucine aminopeptidase (Lap), 6-phosphogluconate dehydrogenase (6-Pgd), phosphoglucose isomerase (Pgi), isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh), adenylate kinase (Adk), and phosphoglucomutase (Pgm). Weight-corrected clearance rates increased significantly (P < 0.05) with increasing multiple-locus heterozygosity and decreased significantly (P < 0.05) with increasing salinity. These data support the idea that heterozygosity-growth correlations may be due in part to differences in clearance rate. However, further study is needed to understand the exact physiological processes which relate heterozygosity and growth.     

Age-dependent changes in rat liver lipid peroxidation and glutathione content induced by acute ethanol ingestion

The study of the influence of the age of animals (13 to 53 weeks) on total liver thiobarbituric acid reactive substances (TBAR) content showed an increase which is maximal in rats of 39 weeks of age compared to young animals (13 weeks), followed by a dimunition in the 53 weeks old group. In this situation, the content of hepatic GSH and total GSH equivalents as well as the GSH/GSSG ratio were decreased with ageing, while GSSG levels were enhanced in the oldest group studied. Acute ethanol intoxication resulted in a marked increase in liver TBAR content in young animals, together with a decline in GSH, total GSH equivalents and GSH/GSSG ratio, and an enhancement in GSSG. These changes elicited by ethanol intake were reduced with ageing. It is concluded that ethanol-induced oxidative stress in the liver is diminished during ageing, despite the progressive decrease in the glutathione content of the tissue observed in control animals.     

Administration of glutathione and lipid peroxidation induced during fasting

It is well known that lipid peroxidation may be initiated or exaggerated by conditions leading to hepatic GSH depletion or altered GSH/GSSG ratio. In our study we evaluated the effects of GSH administration on hepatic, bile and plasma GSH, GSSG and MDA in rats depleted of the tripeptide by a prolonged. fasting. An exteriorized biliary-duodenal fistula was established and GSH or saline solution was administered i.p. for a period of 6h. Rats treated with GSH exhibited an increased GSH and decreased GSSG biliary excretion. Whereas in control rats an opposite pattern was observed, namely enhanced GSSG and decreased GSH biliary excretion. While hepatic GSH and GSSG concentrations were comparable in the two groups, a significant increase in liver and plasma MDA production was found in controls compared to GSH treated rats. Our data suggest a protective role of GSH against the production of lipoperoxidation as evidenced by the decrease of hepatic, biliary and plasma MDA levels and by a decreased percentage of biliary GSSG. In addition, the significant increase of biliary GSH excretion, observed in rats treated with GSH compared to controls, may be due to an increased supply of the tripeptide which is known to be preferentially excreted into bile in the reduced form.     

Glutathione disulfide enhances the reduced glutathione inhibition of lipid peroxidation in rat liver microsomes

Experiments were undertaken to examine the effects of reduced (GSH) and oxidized (GSSG) glutathione on lipid peroxidation of rat liver microsomes. Dependence on microsomal alpha-tocopherol was shown for the GSH inhibition of lipid peroxidation. However, when GSH (5 mM) and GSSG (2.5 mM) were combined in the assay system, inhibition of lipid peroxidation was enhanced markedly over that with GSH alone in microsomes containing alpha-tocopherol. Surprisingly, the synergistic inhibitory effect of GSH and GSSG was also observed for microsomes that were deficient in alpha-tocopherol. These data suggest that there may be more than one factor responsible for the glutathione-dependent inhibition of lipid peroxidation. The first is dependent upon microsomal alpha-tocopherol and likely requires GSH for alpha-tocopherol regeneration from the alpha-tocopheroxyl radical during lipid peroxidation. The second factor appears to be independent of alpha-tocopherol and may involve the reduction of lipid hydroperoxides to their corresponding alcohols. One, or possibly both, of these factors may be activated by GSSG through thiol/disulfide exchange with a protein sulfhydryl moiety.     

Chemically induced antioxidant-sensitive respiration. Relation to glutathione content and lipid peroxidation in the perfused rat liver

The interrelations between the hepatic chemically induced antioxidant-sensitive respiration and the contents of malondialdehyde (MDA) and of reduced glutathione (GSH), were studied in the isolated hemoglobin-free perfused rat liver. Antioxidant-sensitive respiration was induced by the infusion of agents such as ethanol, iron, xanthine or t-butyl hydroperoxide, or by phenylhydrazine pretreatment in vivo. The development of this respiratory component occurred concomitantly with high levels of MDA in the perfused livers, while those of GSH were diminished.     

Protective effect of glutathione and selenium against alloxan induced lipid peroxidation and loss of antioxidant enzymes in erythrocytes

Alloxan is a diabetogenic drug and is known to induce diabetes through generation of free radicals. The toxic oxygen species can be detoxified by antioxidant enzyme system and thus reduce the deleterious effect of lipid peroxidation. Erythrocytes exposed to alloxan induced lipid peroxidationin vivo as well asin vitro. Although alloxan treatment produced a deleterious effect on antioxidant enzymes, pretreatment with glutathione and selenium led to a recovery of the activities of superoxide dismutase and glutathione peroxidase. However, catalase activity increased on alloxan treatment. Alloxan reduced blood glucose level significantly within 60 min but thereafter a slow and steady rise was observed.     

Abundance and ecological significance of the clam Rangia cuneata (Sowerby, 1831) in the upper Barataria Estuary (Louisiana,USA)

We proposed that Rangia cuneata (Sowerby, 1831) is an important estuarine bivalve with ecological significance in three coastal lakes in Barataria Bay, Gulf of Mexico—Lake Cataouatche, Lake Salvador and Lac des Allemands. Our goals were to determine the abundance and distribution of Rangia in these lakes and to measure clearance times to elucidate its potential impacts on phytoplankton communities. The estimated average densities of R. cuneata in Lake Cataouatche, Lake Salvador, and Lac des Allemands were 63, 157, and 107 individuals m⁻², respectively, which is 30% lower than that observed in nearby Lake Pontchartrain. The size of clams in Lake Salvador was between 4 and 50 mm, while individuals in Lake Cataouatche and Lac des Allemands were mostly >20 mm. We postulate that a relatively infrequent large tropical storm transported the larvae from Lake Salvador to the other two lakes 1 year before our sampling to create this size difference. The clams were up to 99.9% of the total benthic biomass in Lake Salvador, 15.9% in Lake Cataouatche, and 40.0% in Lac des Allemands. The R. cuneata biomass values were between 16.2 and 27.6 g m⁻² and the clearance times were 1.0–1.5 days. The clearance times are among the highest previously reported for coastal bivalve communities, which were from cooler climates. The results demonstrate that Rangia can be a critical part of the ecological processes in shallow water systems of the Gulf of Mexico.     

Anaerobic uptake and utilization of glycine-2-14C by the estuarine bivalve Rangia cuneata at three salinities

• 1.1. During facultative anaerobiosis Rangia cuneata acclimated at 5, 10 and 15‰ were injected with 0.1 μCi glycine-2-¹⁴C into the pallial fluid and incubated for 3, 12, 24 and 72 h.
• 2.2. Rangia rapidly accumulated glycine from the pallial fluid.
• 3.3. Accumulation was not significantly different at 5, 10 and 15‰.
• 4.4. Glycine was only metabolized into proteins, and at a slow rate which increased as salinity decreased.
• 5.5. Glycine was not involved in production of ATP during facultative anaerobiosis.
• 6.6. Results suggest both a general decrease in ATP utilization and selective inhibition of certain pathways in order to conserve ATP for the more essential pathways.

     

Glutathione and lipid peroxidation in the aging rat

1. Tissue extracts were prepared from liver, kidney, heart, brain, lung and spleen of male Sprague-Dawley rats of different ages (1-36 months); each of the extracts was analyzed for reduced glutathione (GSH) and lipid peroxides. 2. At all ages the GSH content in the liver was 3-10 times higher than that in other tissues. 3. In the old (36 months) rat the GSH content of all the tissues studied were lower (35-60%) than that in 2.5 month old rat. 4. The lipid peroxides levels increased by age in all tissues studied. 5. These findings indicate that general characteristics of aging tissue may include a decrease in GSH content and increase in lipid peroxides showing a decrease in reducing potential in senescence.     

Effects of t-butyl hydroperoxide on NADPH, glutathione, and the respiratory burst of rat alveolar macrophages

The effects of t-butyl hydroperoxide on glutathione and NADPH and the respiratory burst (an NADPH-dependent function) in rat alveolar macrophages was investigated. Alveolar macrophages were exposed for 15 min to t-butyl hydroperoxide in the presence or absence of added glucose. Cells were then assayed for concanavalin A-stimulated O2 production or for NADPH, NADP, reduced glutathione, glutathione disulfide, glutathione released into the medium and glutathione mixed disulfides. Exposure of rat alveolar macrophages to 1 X 10(-5) M t-butyl hydroperoxide causes a loss of concanavalin A-stimulated superoxide production (the respiratory burst) that can be prevented or reversed by added glucose. Cells incubated without glucose had a higher oxidation state of the NADPH/NADP couple than cells incubated with glucose. With t-butyl hydroperoxide, NADP rose to almost 100% of the NADP + NADPH pool; however, addition of glucose prevented this alteration of the NADPH oxidation state. Cells exposed to 1 X 10(-5) M t-butyl hydroperoxide in the absence of glucose showed a significant increase in the percentage GSSG in the GSH + GSSG pool and increased glutathione mixed disulfides. These changes in glutathione distribution could also be prevented or reversed by glucose. With 1 X 10(-4) M t-butyl hydroperoxide, changes in glutathione oxidation were not prevented by glucose and cells were irreversibly damaged. We conclude that drastic alteration of the NADPH/NADP ratio does not itself reflect toxicity and that significant alteration of glutathione distribution can also be tolerated; however, when oxidative stress exceeds the ability of glucose to prevent alterations in oxidation state, irreversible damage to cell function and structure may occur.     

重金属Cd胁迫对红树蚬的抗氧化酶、消化酶活性和MDA含量的影响

摘要：本文在实验室条件下,观测不同Cd胁迫处理（时间和水平）对红树蚬（Geloina coaxans (Gmelin)）的胃组织中超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性、丙二醛(MDA)含量,及淀粉酶、脂肪酶和蛋白酶等3种消化酶活性的影响效应。结果表明,高浓度组（4.00 mg?L-1和8.00 mg?L-1）在较短的暴露时间即出现SOD 活性显著诱导,而低浓度组则需要更长的暴露时间。所有处理组的CAT活性均先受诱导而后抑制,但去除胁迫后低浓度组活性上升,高浓度组则持续下降。低浓度处理组在胁迫初期MDA含量上升,但随后下降至较低水平;高浓度处理组MDA含量上升稍为滞后,但随后上升至较高水平。在消化酶方面,Cd对红树蚬胃组织淀粉酶的影响基本上表现为抑制;在胁迫初期脂肪酶活性受到显著诱导,随着胁迫时间延长酶活性则下降,同时胁迫解除后不同程度恢复;对蛋白酶的影响效应规律性不明显。显著的效应-剂量间相关关系存在于特定时间的SOD活性（1d）,CAT、淀粉酶活性和MDA含量（7d和恢复6d）。本文还探讨了这些指标作为生物标记物应用于监测海洋重金属污染的可能性。 关键词：红树林;红树蚬;Cd胁迫;消化酶;抗氧化酶;脂质过氧化     

Regulation of lipid peroxidation induced by cumene hydroperoxide in the liver mitochondria of irradiated rats

A study was made of the accumulation of lipoperoxidation products and O2- generation induced by cumene hydroperoxide in mitochondria of irradiated rat liver. O2- generation and formation of lipoperoxidation products were found to be connected with the function of mitochondrial P-450 cytochrome. During the first 24 h following X-irradiation of rats with a dose of 10 Gy, the rate of O2- generation sharply increased and mitochondria could not regulate the intensity of lipoperoxidation with incubation medium tonicity being altered.     

Seasonal variations in the antioxidant defence systems and lipid peroxidation of the digestive gland of mussels

• 1.1. The seasonal variations in the level of antioxidant compounds (glutathione (GSH), vitamin E, carotenoids) and in the activity of antioxidant enzymes, Superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), GSH-peroxidase (EC 1.11.1.9) in the digestive gland of mussels (Mytilus sp.) were evaluated. The lipid peroxidation process was also measured by determining the tissue concentration of malondialdehyde (MDA).
• 2.2. The physiological fluctuations of the antioxidant defence systems were inversely related to the accumulation of lipid peroxidation products (MDA) in the tissue. The observed seasonal variations are presumably related to the changing metabolic status of the animals, itself dependent on such factors as gonad ripening and food availability.
• 3.3. In particular, the obtained data indicate that a reduction of the antioxidant defence systems, occurring during winter, could be directly responsible for an enhanced susceptibility of mussels tissues to oxidative stress, as indicated by the high MDA concentration observed in this period.