Seasonal variation of antioxidant and biotransformation enzymes in barnacle, Balanus balanoides, and their relation with polyaromatic hydrocarbons.

Seasonal variations in the antioxidant enzymes (catalase, superoxide dismutase [SOD], NADH-DT diaphorase), biotransformation enzyme, glutathione-S-transferase (GST) and microsomal lipid peroxidation in digestive tissue of barnacle, Balanus balanoides, from polluted and non-polluted populations have been evaluated. Relationships with accumulated polyaromatic hydrocarbon (PAH) concentration in barnacle tissues and environmental parameters (water temperature, salinity, dissolved oxygen concentration, water pH) were determined. As a general trend, maximum antioxidant enzyme and GST activities were detected in the pre-monsoon period or summer (March-June) followed by a gradual decrease during the monsoon (July October) with a minimum in the post-monsoon period or winter (November February). This pattern was similar to tissue concentrations of PAHs, resulting in a significant positive correlation with antioxidant enzymes, mainly catalase and SOD. Microsomal lipid peroxidation exhibited an almost reverse trend of seasonal variation to that of antioxidant enzyme activities indicating an enhanced susceptibility of barnacle tissues to oxidative stress. Among the environmental parameters, only water temperature seemed to have a significant effect on observed variations of antioxidant enzymes and GST activities. The barnacles from polluted and non-polluted populations exhibited seasonal differences in the activities of all the enzymes studied, particularly catalase, SOD and GST, suggesting the possibility of some biochemical adaptation in organisms from a chronically polluted environment. The results indicated that antioxidant defense components, catalase and SOD, are sensitive parameters that could be useful biomarkers for the evaluation of contaminated aquatic ecosystems. The results also suggested the potentiality of barnacle, B. balanoides, as a bioindicator organism against organic pollution.

Studies on the effects of lipopolysaccharide on lipid peroxidation of erythrocyte and its reversal by mannitol and glycerol.

Gram-negative sepsis often produces endotoxin (LPS) which causes infection. Reduction in tissue perfusion due to microcirculatory failure may lead to septic shock. We studied the effect of LPS on lipid peroxidation of erythrocyte. In vitro studies using 50 microg to 250 microg LPS/ml blood showed increased lipid peroxidation of erythrocyte in a dose-dependent manner. The increased effect of lipid peroxidation does not occur with LPS when erythrocytes were washed to remove plasma and leukocytes. Mannitol and glycerol, known scavengers of hydroxyl radical, arrest the elevation in lipid peroxidation of erythrocytes after LPS treatment. Hemolysis of erythrocytes was reduced with low doses of LPS. Plasma lipid peroxidation was elevated after treatment of blood with LPS. From the results we suggest that the peroxidation of erythrocyte lipid caused by LPS may probably play a role in the production of septic shock.

Antioxidant enzymes in brackishwater oyster, Saccostrea cucullata as potential biomarkers of polyaromatic hydrocarbon pollution in Hooghly Estuary (India): seasonality and its consequences.

Use of antioxidant enzymes as biomarkers often becomes a complicated process at application level because they show considerable seasonal fluctuation due to both natural and biological factors. In this study, we studied the consequences of seasonal variation of antioxidant enzymes [catalase (EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1), glutathione peroxidase (GPX, EC 1.11.1.9) and microsomal NADPH-DT diaphorase (EC 1.6.99.2)] in the digestive gland of wild brackishwatcr oysters, Saccostrea cucullata for biomonitoring against polyaromatic hydrocarbon (PAH) contamination in Hooghly Estuary, north-eastern coast of India. As a general trend, maximum antioxidant enzyme activities were detected in pre-monsoon period or summer (March-June) followed by a gradual decrease during monsoon (July-October) with a minimum in post-monsoon period or winter (November-February) and this pattern was similar to tissue concentrations of PAHs also. The physiological fluctuations of the antioxidant defense systems were inversely-related to the lipid peroxidation indicating an enhanced susceptibility of oyster tissues to oxidative stress during post-monsoon or winter period. However, the oysters from polluted populations exhibited consistent very high PAHs load in their tissues as well as significant increases in the activities of antioxidant enzymes than in non-polluted populations in all three seasons. The results indicated that the antioxidant enzymes, catalase, SOD and microsomal NADPH-DT diaphorase in digestive gland of S. cucullata could be useful biomarkers of PAHs contamination. It also emphasized that seasonal variation of potential biomarkers like such enzymes should be incorporated into interpretation of biomonitoring studies by the use of appropriate controls and identical treatment in analysis of polluted and non-polluted samples.

Protective effect of erythropoietin on the oxidative damage of erythrocyte membrane by hydroxyl radical.

Treatment of red blood cells with the copper (II) ascorbate system causes increased lipid peroxidation, increased membrane microviscosity, and phospholipid translocation with a concurrent decrease in cytosolic catalase and glutathione peroxidase activities. All these changes are prevented if the cells are treated with erythropoietin prior to the exposure to copper (II) ascorbate. The present investigation further indicates that the oxidative damage brought about by copper (II) ascorbate is due to generation of hydroxyl radical and that erythropoietin plays a unique role in protecting the membrane from oxidative damage.

Effect of oxidative stress and erythropoietin on cytoskeletal protein and lipid organization in human erythrocytes.

Phenylhydrazine (PHX)-mediated damage in human red blood cells has been assessed by monitoring the release of tyrosine from cell proteins as well as using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). PHX-treated red blood cells exhibited concentration- and time-dependent tyrosine release. ATP has no effect on the release of tyrosine. This observation is supported by SDS-PAGE pattern of RBC membrane proteins, which shows a correlation between tyrosine release and cytoskeletal protein degradation. PHX requires the presence of erythrocyte cytosolic fraction for the degradation, possibly due to the presence of a proteolytic enzyme in the cytosol. PHX treatment renders the membrane proteins susceptible to the proteolytic attack. Treatment of PHX-exposed erythrocyte with bee venom phospholipase A2 induces the translocation of phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the outer surface of the cell membrane. At the same time, phosphatidylcholine (PC) was translocated towards the inner surface, altering the membrane phospholipid asymmetry. Interestingly, increased tyrosine production followed by translocation of phospholipids across the red blood cell membrane by PHX treatment is completely inhibited by 0.2 units of erythropoietin (EP). Our findings suggest that exposure of red blood cells to an oxidant like PHX causes degradation of cytoskeletal protein by an ATP-independent proteolytic pathway and this in turn allows the transbilayer movement of phospholipids across the cell membrane. EP, by scavenging the hydroxyl radicals produced during interaction of PHX with red blood cells, protects the erythrocytes from oxidative attack.

Effect of endotoxin on protein degradation and lipid peroxidation of erythrocytes.

Sepsis has often been associated with infection due to endotoxin (LPS) produced from gram-negative bacteria. Microcirculatory failure is one of the ultimate causes of septic shock. We studied the effect of endotoxin on the protein breakdown and lipid peroxidation of erythrocyte. In vivo (20 ug LPS/100 g) studies in rats showed increased tyrosine production from erythrocyte, as an index of protein degradation in erythrocyte. In vitro studies using 25 microg to 250 microg LPS per ml also showed similar type of increased effect of endotoxin in protein degradation. Washed erythrocyte devoid of plasma and leucocytes did not show any increased effect after endotoxin treatment. Lipid peroxidation was also increased after endotoxin treatment. However, protein degradation was more prominent than lipid peroxidation. We concluded therefore that the protein degradation and lipid peroxidation of erythrocytes caused by endotoxin are probably related to the production of septic shock.

Effect of cadmium, mercury and copper on partially purified hepatic flavokinase of rat.

The effect of cadmium (Cd2+), mercury (Hg2+) and copper (Cu2+) was studied with partially purified flavokinase (ATP:riboflavin 5'-phosphotransferase EC 2.7.1.26) from rat liver. All the divalent heavy metal cations inhibited flavokinase activity in a concentration-dependent manner. The inhibitory effect of cadmium on the enzyme was completely reversed by increasing concentration, of Zinc (Zn2+) indicating a competition between Zn2+ and Cd2+ for binding with the enzyme. A competition between riboflavin and Cd2+ is also evident from the present investigation. These observations hint at the possibility that Zn2+ and Cd2+ probably compete for the same site on the enzyme where riboflavin binds. However, inhibition of flavokinase by Hg2+ could not be reversed by Zn2+. Our studies further reveal that hepatic flavokinase appears to contain an essential, accessible and functional thiol group(s) which is evident from a concentration dependent inhibition of activity by sulfhydryl reagents like parachloromercuribenzoate (PCMB), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), and N-ethylmaleimide (NEM). Inhibition of flavokinase by sulfhydryl reagents were protected, except in case of NEM inhibition, when the enzyme was incubated with thiol protectors like glutathione (GSH) and dithiothreitol (DTT). Furthermore, the enzyme could also be protected from the inhibitory effect of Cd2+ and Hg2+ by GSH and DTT suggesting that Cd2+ probably interacts with a reactive thiol group at or near the active site of enzyme in bringing about its inhibitory effect.

Effect of cadmium on purified hepatic flavokinase: involvement of reactive -SH group(s) in the inactivation of flavokinase by cadmium.

The effect of cadmium (Cd2+) was studied in vitro on the flavokinase (ATP : riboflavin 5'-phosphotransferase, EC 2.7.1.26) activity purified from rat liver. Cadmium inhibited flavokinase activity in a concentration-dependent manner and the effect was completely reversed by increasing concentration of zinc (Zn2+), indicating a competition between Zn2+ and Cd2+ for binding with the enzyme. Further, a competition between riboflavin and Cd2+ hints at the possibility that Zn2+ and Cd2+ probably compete for the same site on the enzyme where riboflavin binds. Our studies further reveal that hepatic flavokinase contains essential, accessible and functional thiol group(s) as evidenced by a concentration-dependent inhibition by sulfhydryl reagents and protection by thiol protectors like glutathione or dithiothreitol. Furthermore, the enzyme could also be protected from the inhibitory effect of Cd2+ and Hg2+ by glutathione and dithiothreitol suggesting that Cd2+ probably interacts with reactive thiol group at or near the active site of the enzyme to cause inhibition.

Is brain a gluconeogenic organ?

Glucagon increased the activities of alanine amino transferase (AAT), fructose-1:6-bisphosphatase (fru-P2ase) and glucose-6-phosphatase (G-6-Pase) in goat brain tissue by about 100%, 150% and 50% respectively. These increase in activities were reversed by beta-antagonists propranolol. Well known alpha-agonist and antagonist like phenylephrine and phenoxybenzamine also increased AAT and G-6-Pase activities and these increased activities were reversed by propranolol. Phenylephrine and phenoxybenzamine however did not increase brain Fru-P2ase activity. However the most interesting finding is that cerebral cortical slices could produce glucose from alanine and this glucose production was enhanced by glucagon, phenylephrine and phenoxybenzamine. Propranolol reversed the effects of these agonists and antagonist to a great extent. From all these experiments we suggest brain to be a gluconeogenic organ although much less efficient than liver.

Effect of Cu(2+)-ascorbic acid on lipid peroxidation, Mg(2+)-ATPase activity and spectrin of RBC membrane and reversal by erythropoietin.

The effect of erythropoietin (Ep), a glycoprotein hormone, has been studied on lipid peroxidation induced by Cu2+ and ascorbate in vitro, Mg2+ ATPase activity and spectrin of RBC membrane. Our present investigation reveals that Cu2+ and ascorbic acid increases lipid peroxidation of RBC membrane significantly. It has further been observed that under the same experimental condition spectrin, a major cytoskeleton membrane protein, and Mg(2+)-ATPase activity of RBC membrane decrease significantly. However, exogenous administration of Ep completely restores lipid peroxidation and Mg(2+)-ATPase activity and partially recovers spectrin of RBC membrane.

Effect of adrenergic agonists and antagonists on alanine amino transferase, fructose-1:6-bisphosphatase and glucose production in hepatocytes.

Using rat hepatocytes we confirmed our previous results that glucagon and beta-adrenergic agonists increased the enzyme activity of alanine aminotransferase (AAT) and propranolol abolished their effects. Only the enzyme activity was measured and other parameters like quantity of the enzyme or activation due to modification were not looked for. As in perfusion experiment phenylephrine and phenoxybenzamine (alpha-agonist and alpha-antagonist respectively) also alpha-antagonist respectively) also increased the AAT activity in isolated rat hepatocytes and propranolol reversed these effects. The additive effect of glucagon and phenoxybenzamine on AAT was also persistent in hepatocyte system. Fructose-1:6-bisphosphatase (Fru-P2-ase), another key enzyme in gluconeogenic pathway, was elevated by glucagon and other beta-adrenergic agonists both in liver perfusion and isolated hepatocyte experiments and was brought back to the normal level by propranolol. In this case also only the enzyme activity was measured and no other parameters were looked for. Unlike AAT this enzyme was not stimulated by phenylephrine or phenoxybenzamine. But AAT and Fru-P2-ase activities were increased significantly by adenylate cyclase activators like fluoride or forskolin. Thus, it appears that the regulation of fru-P2-ase by glucagon is purely a b-receptor mediated process whereas AAT activation shows a mixed type of regulation where some well known alpha-agonist and antagonists are behaving as beta-agonists. Results further indicate the presence of phosphodiesterase in hepatocyte membrane which was stimulated by glucagon and brought back to the normal level by propranolol. The different adrenergic compounds stated above, not only modified the activity of the above two enzymes but also stimulated glucose production by hepatocytes from alanine which was in turn abolished by propranolol as well as amino oxyacetate (AOA), a highly specified inhibitor of AAT. This confirm the participation of AAT in gluconeogenesis from alanine in liver. Forskolin and fluoride also increased the glucose production from alanine and showed additive effects with glucagon, phenylephrine and phenoxybenzamine.

Effect of glucagon and some other alpha and beta adrenergic agonists and antagonists on alanine amino transferase of perfused rat liver.

Glucagon increased alanine amino transferase (AAT) activity in perfused rat liver by about 90% over control. Propranolol, the beta receptor antagonist, abolished the effect of glucagon on this enzyme. Well known beta receptor agonists like isoproterenol, norepinephrine and epinephrine also increased the enzyme activity under identical condition and the enhancement was similarly abolished by propranolol. These experiments suggest that the effect of glucagon on AAT was mediated through beta adrenergic receptor. However, the interesting observation was that phenylephrine, alpha receptor agonist and phenoxybenzamine and tolazoline, two alpha receptor antagonists, increased the AAT activity like glucagon in perfusion experiments and the effects of all these three agents were also abolished by propranolol. Glucagon, when perfused with phenoxybenzamine showed some additive effect. From all these results we are proposing that in our system phenoxybenzamine is acting as beta agonist although it is known to be an alpha antagonist.

Effect of erythropoietin on membrane lipid peroxidation, superoxide dismutase, catalase, and glutathione peroxidase of rat RBC.

Starved animals having low levels of erythropoietin in blood showed increased MDA, fluorescent pigments, and met-Hb values whereas the hemoglobin concentration decreased significantly on starvation. In vivo and in vitro studies with Ep reversed the effects of starvation and brought these values close to normal. The activities of the enzymes (SOD, catalase, GSH-PX, GR G6PD, and 6PGD) which protect the RBC membrane directly or indirectly from peroxidative threat, decreased on starvation and restored to normal levels after Ep treatment.

Effect of erythropoietin on the glucose transport of rat erythrocytes and bone marrow cells.

The effect of Ep on radioactive glucose and methyl-alpha-D-glucoside transport by rat erythrocytes and bone marrow cells were studied. There is initial linearity followed by saturation kinetics of [14C]glucose transport by the erythrocytes of starved and starved plus Ep-treated rats at different concentrations of glucose. Starvation caused slight inhibition of glucose transport which increased markedly on Ep administration to starved rats. Normal animals failed to show any significant change in glucose transport after Ep treatment. Methyl-alpha-D-glucoside inhibited the Ep-stimulated glucose transport significantly. Ep also stimulated the transport of radioactive methyl-alpha-D-glucoside which was competitively inhibited in presence of D-glucose. Glucose transport in erythrocytes was found to be sensitive to metabolic inhibitors like azide and DNP. A sulfhydryl reagent and ouabain also inhibited the transport process. Ep stimulated glucose and methyl-alpha-D-glucoside transport in the bone marrow cells of starved rats. The sugar analog competitively inhibited the glucose transport in bone marrow cells and vice versa.

The effect of submaxillariectomy on the uterine peroxidase activity and [14C]phenylalanine incorporation into uterine protein in rats.

Surgical removal of submaxillary glands in immature female rats caused an increase in size and about 3-fold increase in dry and wet weight of the uterus compared to that of the sham operated animals of the same age group. Histological examination reveal a significant increase in the diameter of the uterus with considerable elongation of the luminal epithelium from cubical to columnar in the experimental group. Biochemical studies showed that the uterine peroxidase, a marker enzyme for oestrogen action, increased (P less than 0.01) on submaxillariectomy. Incorporation of [14C]phenylalanine into the nuclear fraction of uterus was also enhanced significantly on removal of submaxillary glands. The elevation of peroxidase activity as well as of [14C]phenylalanine incorporation into the nuclear fraction due to removal of submaxillary glands were abolished in ovariectomized rats suggesting the involvement of ovarian hormones. The results show that oestrogen was responsible for all the above mentioned changes, whereas progesterone had little effect. Results further suggest the existence of a factor in the submaxillary glands through which they exert an effect on the uterus and ovary.

Effect of cold exposure on the amylase activity of submaxillary gland of rats.

Cold exposure of rats for 3 h (6 +/- 2 degrees C) caused an increase in amylase activity of the submaxillary gland. This effect was not observed in other salivary glands i.e. parotid and sublingual or in the pancreas. The increase of amylase activity during cold exposure was completely abolished by the beta-receptor antagonist, propranolol, and the alpha-receptor antagonist, phenoxybenzamine, reduced the effect. Administration of actinomycin D to the cold-exposed rats produced a tremendous increase of enzyme activity instead of abolition of the increase as had been expected.

Effect of erythropoietin on the different ATPases and acetylcholinesterase of rat RBC membrane.

The effect of Ep on different ATPases and acetylcholinesterase of rat RBC membrane was studied. Starvation caused a slight decrease in Mg2+-, Ca2+-, and Na+ + K+-ATPases. However, these enzyme activities were markedly increased on Ep treatment of starved rats. Specific activities of all three ATPases increased linearly with increasing concentration of Ep. Under identical conditions the hormone failed to stimulate the ATPase activity of liver plasma membrane. Desensitization by fluoride of allosteric inhibition of erythrocyte membrane-bound Na+ + K+-ATPase was observed under starvation which showed a return to normal n values on Ep administration. The enzyme from normal animals was inhibited almost completely at 0.1 mM fluoride whereas enzyme from starved and Ep-treated animals showed only about 50% inhibition at that fluoride concentration. Ep increased the acetylcholinesterase activity of normal RBC membrane to a small extent whereas the stimulation was much higher under starvation. The fluoride inhibition curve of this enzyme changed from sigmoidal to hyperbolic under starvation which again changed to allosteric on administration of Ep. These changes were closely correlated to n values. Red blood cells of Ep-treated animals became more susceptible to osmotic shock under the experimental conditions.

Horseradish peroxidase-catalyzed conversion of iodine to iodide in presence of EDTA and H2O2.

EDTA (4 mM) blocks the oxidation of iodide to I-3 (increase of extinction at 353 nm) by H2O2 catalyzed by horseradish peroxidase, which is reversed by the addition of an equimolar concentration of Zn2+. Addition of suboptimal concentration of EDTA (2 mM) not only decreases the rate of forward reaction of I-3 formation but also causes loss of extinction of the same when I-3 is generated. The loss of extinction of I-3 is proportional to the enzyme concentration and is blocked by azide, the inhibitor of the peroxidase. EDTA also causes bleaching of nonenzymatically formed I-3 (from iodide and H2O2) only in the presence of horseradish peroxidase, and the effect is reversed by the equimolar concentration of Zn2+. Both the bleaching of I-3 by EDTA and reversal of EDTA effect by Zn2+ are sensitive to azide. The decrease of extinction of I-3 (formed by dissolving iodine in KI solution) is dependent on EDTA, H2O2, and horseradish peroxidase. Molecular iodine is also bleached but at a slower rate than I-3. Evidence is presented to show that this bleaching of I-3 is due to enzymatic conversion of I-3 to iodide in presence of EDTA and H2O2 and this involves pseudocatalatic degradation of H2O2 to O2.