Release of mercury from dental amalgam and its influence on salivary antioxidant activity.

Dental amalgam fillings are known to release significant amounts of mercury (Hg) in saliva which could represent a continuous source of oxidative damage to mouth tissues. The present investigation was aimed at verifying this hypothesis by determining a possible correlation between salivary Hg levels and salivary total antioxidant activity (TAA), which is used as an index of oxidative stress. Samples of saliva from 34 healthy donors were analyzed for Hg content, by vapor atomic absorption spectrometry, and for TAA, by determining the ferric reducing ability ('FRAP' method). A significant correlation between Hg and the number of amalgam restorations or total amalgam surface was evident in both the male and female subjects. A significant negative correlation between TAA and Hg levels or number of amalgam restorations or amalgam surface was evident in females, indicating that small increases in salivary Hg were sufficient to produce a decrease in salivary TAA. On the other hand, no significant correlation was found in the males. The present study provides, for the first time, evidence of a pro-oxidant role of the amalgam Hg chronically released in saliva.

Determination of a redox compensation index and its relationships to glycaemic control in type 2 diabetes mellitus.

The measurement of single parameters of oxidative stress in biological fluids can often give results difficult to interpret as to the real involvement of oxidative processes in a given disease condition. In the present study we propose a novel integrated parameter, called "redox compensation index", obtained by combining the results of two established and convenient procedures, i.e. the Fox-2 assay for plasma lipid hydroperoxides and the ferric reducing/antioxidant power (FRAP) assay for total antioxidant potential of plasma. These procedures were employed for the evaluation of oxidative stress in a group of patients with type 2 diabetes mellitus, a condition in which oxidative processes are implicated in the development of complications. In type 2 diabetic patients, plasma lipid hydroperoxides were directly correlated with levels of glycated hemoglobin. On the other hand, a significant inverse correlation was observed between levels of glycated hemoglobin and redox compensation values. The data reported suggest that the redox compensation index could represent a convenient parameter for the direct appraisal of oxidative status in clinical subjects, and are in support of the proposed role of protein glycation in production of oxidative alterations during type 2 diabetes mellitus.

Release of mercury from dental amalgam and its influence on salivary antioxidant activity.

Dental amalgam fillings are known to release significant levels of mercury (Hg) in saliva which could represent a continuous source of oxidative damage to tissues. The present investigation was aimed at verifying this hypothesis by determining a possible correlation between salivary Hg levels and salivary total antioxidant activity (TAA), used as an index of oxidative stress. Samples of saliva from 34 healthy donors were analyzed for Hg content, through vapor atomic absorption spectrometry, and for TAA, by determining the ferric reducing ability ('FRAP' method). A significant correlation between Hg and the number of amalgam restorations or total amalgam surface was evident in both the male and female subjects. A significant negative correlation between TAA and Hg levels or number of amalgam restorations or amalgam surface was evident in females, indicating that small increases in salivary Hg were sufficient to produce a decrease in salivary TAA. On the other hand, no significant correlation was found in the males. The present study provides, for the first time, evidence of a pro-oxidant role of the amalgam Hg chronically released in saliva.

Protection against oxidative damage of erythrocyte membrane by the flavonoid quercetin and its relation to iron chelating activity.

Incubation of glutathione (GSH) depleted mouse erythrocytes with the oxidants phenylhydrazine, acrolein, divicine and isouramil resulted in the release of free iron and in lipid peroxidation and hemolysis. The addition of the flavonoid quercetin, which chelates iron and penetrates erythrocytes, resulted in remarkable protection against lipid peroxidation and hemolysis. The protection seems to be due to intracellular chelation of iron, since a semi-stoichiometric ratio between released iron and the amount of quercetin necessary to prevent lipid peroxidation and hemolysis was found. Incubation of GSH depleted human erythrocytes with divicine and isouramil did not induce lipid peroxidation and hemolysis in spite of a substantial release of iron. However, divicine and isouramil produced alterations of membrane proteins, such as spectrin and band 3, as well as formation of senescent cell antigen. The addition of quercetin prevented these alterations.

Release of free, redox-active iron in the liver and DNA oxidative damage following phenylhydrazine intoxication.

Following the subchronic intoxication of rats with phenylhydrazine, resulting in marked anemia, reticulocytosis, methemoglobinemia and increased hemocatheresis, the hepatic content of total iron was increased, as was hepatic ferritin and its saturation by iron. A striking increase (approximately 7-fold) was also observed in free iron which appeared to be redox-active. The increase in liver free iron involved the hepatocellular component of the liver. Since DNA is one of the cellular targets of redox active iron, liver DNA from phenylhydrazine-treated rats was analyzed by electrophoresis and found to be markedly fragmented. Experiments with isolated hepatocytes in culture or in suspension challenged with phenylhydrazine or Fe-nitrilotriacetate strongly suggested that the DNA damage was due to reactive iron rather than to the hepatic metabolism of phenylhydrazine. The levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a specific marker of oxidative DNA damage, were significantly higher in phenylhydrazine-treated rats as compared to untreated controls. The prolongation of phenylhydrazine treatment over a period of 6 weeks resulted in a persistent damage to DNA and in phenotypic changes such as an increase in hepatocyte gamma-glutamyl transpeptidase (gamma-GT, EC 2.3.2.2) activity. Possible relationships between iron overload, iron release, DNA damage and tumor initiation are discussed.

Iron release, membrane protein oxidation and erythrocyte ageing.

The aerobic incubation of erythrocytes in phosphate buffer for 24-60 h (a model of rapid in vitro ageing) induced progressive iron release and methemoglobin formation. Membrane proteins showed electrophoretic alterations and increase in carbonyl groups (as documented by IR spectroscopy). None of these phenomena were seen when the erythrocytes were incubated under anaerobic conditions. The membranes from aerobically incubated cells bound a much higher amount of autologous IgG than those from anaerobically incubated ones, suggesting that the aerobic incubation gives rise to the senescent antigen. The addition of ferrozine during the aerobic incubation prevented both the IgG binding and the protein alterations seen in the IR spectra, suggesting an intracellular chelation of the released iron by ferrozine.

Purification of NADPH-dependent dehydroascorbate reductase from rat liver and its identification with 3 alpha-hydroxysteroid dehydrogenase.

Rat liver cytosol has been found to reduce dehydroascorbic acid (DHAA) to ascorbic acid in the presence of NADPH. The enzyme responsible for such activity has been purified by ammonium sulphate fractionation, DEAE-Sepharose, Sephadex G-100 SF and Reactive Red column chromatography, with an overall recovery of 27%. SDS/PAGE of the purified enzyme showed one single protein band with an M(r) of 37,500. A similar value (36,800) was found by gel filtration on a Sephadex G-100 SF column. The results indicate that the enzyme is a homogeneous monomer. The Km for DHAA was 4.6 mM and the Vmax. was 1.55 units/mg of protein; for NADPH Km and Vmax. were 4.3 microM and 1.10 units/mg of protein respectively. The optimum pH was around 6.2. Several typical substrates and inhibitors of the aldo-keto reductase superfamily have been tested. The strong inhibition of DHAA reductase effected by steroidal and non-steroidal anti-inflammatory drugs, together with the ability to reduce 5 alpha-androstane-3,17-dione strongly, suggest the possibility that DHAA reductase corresponds to 3 alpha-hydroxysteroid dehydrogenase. Microsequence analysis performed on the electro-transferred enzyme band shows that the N-terminus is blocked. Internal primary structure data were obtained from CNBr-derived fragments and definitely proved the identity of NADPH-dependent DHAA reductase with 3 alpha-hydroxysteroid dehydrogenase.

Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver.

GSH-dependent enzymic reduction of dehydroascorbic acid to ascorbic acid has been studied in rat liver cytosol. After gel filtration of cytosol on Sephadex G-100 SF, dehydroascorbate reductase activity was recovered in two distinct peaks, one corresponding to glutaredoxin (an enzyme already known for its dehydroascorbate reductase activity) and another, much larger one, corresponding to a novel enzyme different from glutaredoxin. The latter was purified to apparent homogeneity. The purification process involved (NH4)2SO4 fractionation, followed by DEAE-Sepharose, Sephadex G-100 SF and Reactive Red chromatography. SDS/PAGE of the purified enzyme in either the presence or absence of 2-mercaptoethanol demonstrated a single protein band of M(r) 31,000. The M(r) determined by both Sephadex G-100 SF chromatography and h.p.l.c. was found to be approx. 48,000. H.p.l.c. of the denatured enzyme gave an M(r) value identical with that obtained by SDS/PAGE (31,000). The apparent Km for dehydroascorbate was 245 microM and the Vmax. was 1.9 mumol/min per mg of protein; for GSH they were 2.8 mM and 4.5 mumol/min per mg of protein respectively. The optimal pH range was 7.5-8.0. Microsequence analysis of the electro-transferred enzyme band showed that the N-terminus is blocked. Data on internal primary structure were obtained from CNBr-and N-chlorosuccinimide-derived fragments. No significative sequence similarity was found to any of the protein sequences contained in the Protein Identification Resource database.

Protection by ascorbic acid against oxidative injury of isolated hepatocytes.

1. The ability of ascorbic acid to protect from prooxidant-induced toxic injury was investigated in isolated, intact rat hepatocytes, whose ascorbic acid content had been restored by means of exogenous supplementation. 2. Ascorbate-supplemented and ascorbate-non-supplemented cells in suspension were treated with a series of different prooxidants (allyl alcohol, diethyl maleate, carbon tetrachloride, menadione), and the development of lipid peroxidation and cell injury was evaluated. 3. With allyl alcohol and diethyl maleate, ascorbic acid was able to protect cells from both lipid peroxidation and cell injury. The same protection was offered by ascorbate also in hepatocytes obtained from vitamin E-deficient animals. 4. With carbon tetrachloride, ascorbate supplementation did not affect the initial steps of lipid peroxidation, but nevertheless provided a marked protection against lipid peroxidation and cell injury at later times of incubation. The protection was unaffected by the vitamin E content of cells. 5. With menadione, a toxin which does not induce lipid peroxidation, ascorbic acid did not protect cells against injury. 6. It is concluded that ascorbic acid can act as an efficient antioxidant in isolated rat liver cells, with protection against cell injury. The antioxidant effect appears primarily to involve membrane lipids, and can be independent from the cellular content of vitamin E, thus suggesting that ascorbic acid can play a direct and independent role in the intact cell, in addition to its synergistic interaction with vitamin E described in other models.