Cesare Sblano

Molecular inhibitory mechanisms of antioxidant enzymes in rat liver and kidney by cadmium

Catalase, Mn-superoxide dismutase (MnSOD) and Cu,Zn-superoxide dismutase (CuZnSOD) activities were studied in rat liver and kidney 6-48 h after CdCl(2) intraperitoneal administration or 10-30 days daily oral CdCl(2) intake in drinking water. This approach provided some indications as to the sensitivity of each enzyme to cadmium toxicity. These experiments showed that the formation of thiobarbituric acid reactive substance (TBARS) did not strictly depend on how well the antioxidant enzyme worked. From in vitro experiments it appeared that TBARS removal by vitamin E did not restore the three enzyme activities at all. As for cadmiums inhibitory mechanism on catalase activity, our data, obtained in the pH range 6.0-8.0, are a preliminary indication that the negative effect of this metal is probably due to imidazole residue binding of His-74 which is essential in the decomposition of hydrogen peroxide. Cadmium inhibition of liver mitochondrial MnSOD activity was completely removed by Mn(2+) ions, suggesting that the reducing effect on this enzyme is probably due to the substitution of cadmium for manganese. We also observed the antioxidant capacity of Mn(2+) ions, since they were able to normalize the increased TBARS levels occurring when liver mitochondria were exposed to cadmium. The reduced activity of CuZnSOD does not seem to be due to the replacement of Zn by Cd, nor to the peroxides formed. As this enzyme activity was almost completely recovered after 48 h, we hypothesize that the momentary inhibition is imputable to a cadmium/enzyme interaction. This causes some perturbation in the enzyme topography which is critical for its catalytic activity. The pathological implications linked to antioxidant enzyme disorders induced by cadmium toxicity are discussed.

The response of rat liver lipid peroxidation, antioxidant enzyme activities and glutathione concentration to the thyroid hormone.

1. In liver microsomes from hyperthyroid rats NADPH-dependent lipid peroxidation induces a hydroperoxide formation 56% higher than that in euthyroid ones. 2. The addition of 5 microM Fe2+ (or Fe3+) strongly decreases the hydroperoxide level in favour of that of TBA-reactive substances. Higher iron concentrations (30 microM) have no significant effect. 3. In hepatocytes from hyperthyroid rats CCl4-induced lipid peroxidation produces an amount of TBA-reactive substances four times higher than that in those from euthyroid rats. 4. In the liver of hyperthyroid rats a GSH concentration decrease (by about 35%) is found while the opposite occurs in the blood of the same animals where GSH increases 2.5 times. 5. It is shown that in the liver of hyperthyroid rats, besides higher lipid peroxidation, a more active defense mechanism is operating since both glutathione peroxidase and glutathione reductase specific activities are higher than in euthyroid rats.

Antioxidant effect of hydroxytyrosol (DPE) and Mn2+ in liver of cadmium-intoxicated rats.

Liver TBARS formation in cadmium-intoxicated rats was completely reduced by administering a low amount of MnCl(2) (2 mg/kg b.w.) 1 h before intoxication. A similar antioxidant effect was first shown by hydroxytyrosol (2-(3,4-dihydroxyphenyl)ethanol, (DPE), a phenolic compound present in olive oil, given twice to rats (9 mg/kg b.w.) after cadmium administration. The antioxidant properties shown in vivo by both Mn(2+) and DPE were also active in vitro when rat liver microsomes were subjected to lipid peroxidation by cadmium or other prooxidant systems. The increase in liver glutathione concentrations occurring in cadmium-intoxicated rats, was also found, for the first time, 24 h after MnCl(2) administration. Unlike cadmium intoxication, which caused a higher formation of both glutathione and TBARS, Mn(2+) induced glutathione synthesis without any TBARS formation. The same situation was also observed when cadmium plus Mn(2+) or cadmium plus DPE was given to rats. Our data show that: (a). both DPE and low Mn(2+) concentrations may have an antioxidant effect in the livers of cadmium-intoxicated rats and (b). Mn(2+), like cadmium, induces liver glutathione synthesis and this effect is probably independent of TBARS formation.

The Nrf2 transcription factor contributes to the induction of alpha-class GST isoenzymes in liver of acute cadmium or manganese intoxicated rats: comparison with the toxic effect on NAD(P)H:quinone reductase.

In rat liver, in addition to their intrinsic transferase activity, alpha-class GSTs have Se-independent glutathione peroxidase activity toward fatty acid hydroperoxides, cumene hydroperoxide and phospholipids hydroperoxides but not toward H(2)O(2.) We have previously shown that hepatic GST activity by these isoenzymes is significantly increased 24h after cadmium or manganese administration (Casalino et al., 2004). Here it is reported that Se-independent glutathione peroxidase activity by alpha-class GSTs is also stimulated in the liver of intoxicated rats. The stimulation is associated with a higher level of alpha-class GST proteins, whose induction is blocked by actinomycin D co-administration. The observed Se-independent glutathione peroxidase activity is due to alpha-class GST isoenzymes, as indicated by the studies with diethyldithiocarbamate which, at any concentration, equally inhibits both GST and Se-independent glutathione peroxidase and is an uncompetitive inhibitor of both enzymes. As for liver Se-GSPx, it is not at all affected under these toxic conditions. For comparison, we have evaluated the status of another important antioxidant enzyme, NAD(P)H:quinone reductase, 24h after cadmium or manganese administration. NQO1 too results strongly stimulated in the liver of the intoxicated rats. In these animals, a higher expression of Nrf2 protein is observed, actively translocated from the cytoplasm to the nucleus. The results with the transcription inhibitor, actinomycin D, and the effects on Nrf2 protein are the first clear indication that acute manganese intoxication, similarly to that of cadmium and other heavy metals, increases both the hepatic level of Nrf2 and its transfer from the cytoplasm to the nucleus where it actively regulates the induction of phase II enzymes.

A possible mechanism for initiation of lipid peroxidation by ascorbate in rat liver microsomes.

The mechanism by which lipid peroxidation progresses has been known for years, but there is disagreement regarding the mode of its initiation. The aim of this study was to examine: (a) the role of endogenous iron in the initiation of ascorbate-induced lipid peroxidation in microsomal and liposomal membranes; (b) the role of oxygen-free radicals in this process; and (c) the redox state of ascorbate during the course of lipid peroxidation. Ascorbate-induced lipid peroxidation was assessed by measuring hydroperoxide and thiobarbituric acid reactive substances (TBARS) formation in membranes after incubation in Tris-HCl buffer (pH 7.4) for 15 min. To confirm the role of endogenous iron and oxygen-free radicals, the effect of iron chelating agents (EDTA and thiourea) and radical scavengers (benzoate, mannitol, catalase and SOD) on lipid peroxidation was examined. Spectrophotometric measurements and ESR spectra have made it possible to determine ascorbate concentration and its redox state. Ascorbate promoted lipid peroxidation in both rat liver microsomes and liposomes without addition of exogenous iron. Iron chelating agents such as EDTA and thiourea inhibited lipid peroxidation, while SOD, catalase, mannitol and benzoate had no effect. The addition of 5 microM Fe2+ (or Fe3+) to the incubation mixture did not significantly alter hydroperoxide production, but that of TBARS was increased. Lipid peroxidation significantly altered the fatty acid profile in microsomes and liposomes, the most affected being the C20:4 and C22:6 species. Ascorbate in Tris-HCl buffer (pH 7.4) autoxidized very slowly. Its oxidation was catalyzed by Fe3+ ions at a rate determined by incubation time and iron concentration. In contrast, no ascorbate oxidation occurred in the presence of microsomes when lipid peroxidation was proceeding at a maximal rate. Under these conditions a typical ascorbyl radical ESR spectrum signal greater than that arising from ascorbate alone was obtained and the magnitude of this signal was unchanged by variations of microsome or ascorbate concentrations. A ferrous ion ascorbyl radical complex was responsible for this signal. These results suggest that an ascorbate-microsomal iron complex is responsible for the initiation of lipid peroxidation, and that during this process ascorbate remains in its reduced form.

Studies on the Effect of Salts on the Channel Activity of Kissper, a Kiwi Fruit Peptide

Fruits and vegetables are known to possess health benefits: indeed, they contain a large number of bioactive molecules with a positive impact on human health. Dietary proteins possess specific biological properties which make these components potential ingredients of functional or health-promoting foods. Many of these properties are attributed to physiologically-active peptides encrypted in protein molecules. Kissper, a 39-residue peptide isolated from kiwi fruit (Actinidia deliciosa), derives from the processing of kiwellin, a well-represented novel allergenic protein present in the edible part of the fruit. Kissper has six cysteine residues forming three disulfide bridges. This cysteine pattern is similar, but not identical, to those observed in some plant and animal proteins, including toxins involved in defence mechanisms. We show that kissper forms ion channels in DOPS:DOPE:POPC PLMs in different media, such as KCl, potassium gluconate, potassium citrate and potassium phosphate monobasic. Kisspers channels show a strong voltage dependence in all media used, and an estimated pore diameter in the range from 1.9 to 7.6 A in KCl medium. Finally, kisspers pores shift selectivity from anions to neutral to cations, depending on the nature of the salt solutions. Therefore we propose the kissper as a novel nutraceutical component of kiwi fruit that may have a positive impact on human health due to its channel-like pathways which modulate membrane permeability. The kissper action might promote the adsorption of substances, in particular, within the gastrointestinal tract, helping digestion, preventing constipation and harmful accumulation of metabolites.

Effects of n-Octyl-β-D-Glucopyranoside on Human and Rat Erythrocyte Membrane Stability Against Hemolysis

The practical importance for the pharmaceutical and cosmetics industries of the interactions between biological membranes and surfactant molecules has led to intensive research within this area. The interactions of non-ionic surfactant n-octyl-β-D-glucopyranoside (OG) with the human and rat erythrocyte membranes were studied. The in vitro hemolytic and antihemolytic activities were determined by employing a method in which both erythrocytes were added to the hypotonic medium containing OG at different concentrations, and the amount of haemoglobin released was determined. n- octyl-β-D-glucopyranoside was found to have a biphasic effect on both types of erythrocyte membrane. We also investigated the interactions of OG with the erythrocyte membrane in isotonic medium; the dose-dependent curves show similar behaviour in both human and rat erythrocytes. Our results showed that OG has greater antihemolytic potency on rat than on human erythrocytes; furthermore, rat erythrocytes were more sensitive than human erythrocytes to hypotonic shock. How the different lipoprotein structure of these erythrocytes determines a difference in antihemolytic activity is discussed.