Evidence of cadmium and mercury involvement in the Aß42 aggregation process.

Aß42 is a small peptide formed from 42 aminoacids that presents a great propensity to aggregate until it forms fibrils. Aß42 aggregation and fibrillation are very complex processes whose molecular mechanisms seem to depend on characteristics intrinsic to the peptide molecule, as well as extrinsic factors. Peptide concentration, mean pH and several substances, including metal ions, are principal extrinsic factors for the oligomerization process. Different metals affect the aggregation of the Aß42 molecule, and their toxicity favours the misfolding and aggregation of the peptide. In this study, we evaluate the effect of different concentrations of Cd2+ and Hg2+ on the Aß42 peptide in solution by different methods. The toxicity of Aß42 was evaluated with the MTT assay, while the aggregation process was monitored by single-channel measurements, electrophoresis and western blot. Cd2+ and Hg2+ seem to favour the formation of high-molecular-weight aggregates, to decrease ion channel turnover inside the membrane and to significantly increase Aß42 toxicity.

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.

Acute cadmium intoxication induces alpha-class glutathione S-transferase protein synthesis and enzyme activity in rat liver.

Acute cadmium intoxication affects glutathione S-transferase (GST) in rat liver. It has been found that 24h after i.p. cadmium administration to rats, at a dose of 2.5 mg CdCl(2)kg(-1) body weight, the activity of this enzyme in liver cytosol increased by 40%. A less stimulatory effect persisted till 48 h and thereafter the enzyme activity normalized. Since, GST isoenzymes belong to different classes in mammalian tissues, we used quantitative immunoassays to verify which family of GST isoenzymes is influenced by this intoxication. Only alpha-class glutathione S-transferase (alpha-GST) proteins were detected in rat liver cytosol and their level increased by about 25%, 24h after cadmium treatment. No pi-GST isoforms were found in liver cytosol from either normal or cadmium-treated rats. Co-administration of actinomycin D with cadmium normalized both the protein level and the activity of alpha-GST, suggesting that some effect occurs on enzyme transcription of these isoenzymes by this metal. On the other hand, it seems unlikely that the stimulatory effect is due to the high level of peroxides caused by lipid peroxidation, since Vitamin E administration strongly reduced the TBARS level, but did not cause any GST activity decrease.

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 cadmium's 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.

Rat liver glutathione S-transferase activity stimulation following acute cadmium or manganese intoxication.

The effect of cadmium or manganese administration on rat liver glutathione S-transferase (GST) has been investigated. The activity of this enzyme in liver cytosol, where almost all the cellular activity is present, had increased by more than 36% 24 h after a single i.p. injection of CdCl(2) (2.5 mg kg(-1) b.w.) or MnCl(2) (2.0 mg kg(-1) b.w.). After shorter and longer time intervals, a lower enzyme activity stimulation was observed in both cases. When liver cytosol was incubated for 10 min with 75 microM CdCl(2) or 40 microM MnCl(2), no effect was observed on enzyme activity. The increase in GST following cadmium or manganese administration was blocked by prior administration of actinomycin D, indicative of a possible transcription-dependent response. The liver soluble GST from both control and metal-treated rats was not at all affected by Vitamin E, in the range of 20-300 microM. By contrast, hematin was seen to be a competitive inhibitor of this liver enzyme from both types of rats by using CDNB as substrate and the K(i) value was equal to 0.22 microM. The possibility that under the conditions used class alpha GST isoenzymes are affected by cadmium or manganese is discussed.

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.