Immuno-spin trapping detection of antioxidant/pro-oxidant properties of zinc or selenium on DNA and protein radical formation via hydrogen peroxide.

Trace elements can participate in the catalysis of group-transfer reactions and can serve as their structural components. However, most of them including zinc and selenium have multifunctional roles in biological environments such as antioxidant and/or pro-oxidant effects, as concentration-dependent manner. Although it has been demonstrated the antioxidant actions of either selenium or zinc compounds, there are several documents pointing out their pro-oxidant/oxidant roles in biological systems. Here we have used ELISA-based immuno-spin trapping, a method for detection of free radical formation, to detect whether or not a zinc compound, Zn3(PO4)2, or a selenium compound, Na2SeO3, has antioxidant and/or pro-oxidant effect on 5,5-Dimethyl-1-Pyrroline-N-Oxide (DMPO)-DNA nitrone adducts induced with Cu(II)-H2O2-oxidizing system in in vitro preparations. Second, we examined whether this technique is capable to demonstrate the different DMPO-protein nitrone adduct productions in isolated protein crude of hearts from normal rats (CON) or rats with metabolic syndrome (MetS). Our data demonstrated that either Zn(2+) (100 µM) or SeO3(-2) (50 nM) has very strong antioxidant action against 200 µM H2O2-induced DMPO-DNA nitrone adduct production, whereas their higher concentrations have apparent pro-oxidant actions. We also used verification by Western blotting analysis whether immuno-spin trapping can be used to assess H2O2-induced DMPO-protein nitrone adducts in heart protein crudes. Our Western blot data further confirmed the ELISA-data from proteins and demonstrated how Zn(2+) or SeO3(-2) are dual-functioning ions such as antioxidant at lower concentrations while pro-oxidant at higher concentrations. Particularly, our present data with SeO3(-2) in DMPO-protein nitrone adducts, being in line with our previous observation on its dual-actions in ischemia/reperfusion-induced damaged heart, have shown that this ion has higher pro-oxidant actions over 50 nM in MetS-group compared to that of CON group.

Beta-blocker timolol alleviates hyperglycemia-induced cardiac damage via inhibition of endoplasmic reticulum stress.

Current data support that pharmacological modulators of endoplasmic reticulum stress (ERS) have therapeutic potential for diabetic individuals. Therefore, we aimed to examine whether timolol, having free radical-scavenger action, besides being a ß-blocker, exerts a cardioprotective effect via inhibition of ERS response in diabetic rats in a comparison with an antioxidant N-acetylcysteine (NAC). Histopathological data showed that either timolol- or NAC-treatment of diabetic rats prevented the changes in mitochondria and nucleus of the cardiac tissue while they enhanced the cellular redox-state in heart as well. The levels of ER-targeted cytoprotective chaperones GRP78 and calnexin, unfolded protein response signaling protein CHO/Gadd153 besides the levels of calpain, BCL-2, phospho-Akt, PUMA, and PML in the hearts from diabetic rats, treated with either timolol or NAC, are found to be similar among these groups, although all these parameters were markedly preserved in the untreated diabetics compared to those of the controls. Taken into consideration how important a balanced-ratio between anti-apoptotic and pro-apoptotic proteins for the maintenance mitochondria/ER function, our results suggest that ERS in diabetic rat heart is mediated by increased oxidative damage, which in turn triggers cardiac dysfunction. Moreover, we also demonstrated that timolol treatment of diabetic rats, similar to NAC treatment, induced a well-controlled redox-state and apoptosis in cardiac myocardium. We, thus for the first time, report that cardioprotective effect of timolol seems to be associated with normalization of ER function due to its antioxidant action in cardiomyocytes even under hyperglycemia.

Enhancement of cellular antioxidant-defence preserves diastolic dysfunction via regulation of both diastolic Zn2+ and Ca2+ and prevention of RyR2-leak in hyperglycemic cardiomyocytes.

We examined whether cellular antioxidant-defence enhancement preserves diastolic dysfunction via regulation of both diastolic intracellular free Zn(2+) and Ca(2+) levels ([Zn(2+)]i and [Ca(2+)]i) levels N-acetyl cysteine (NAC) treatment (4 weeks) of diabetic rats preserved altered cellular redox state and also prevented diabetes-induced tissue damage and diastolic dysfunction with marked normalizations in the resting [Zn(2+)]i and [Ca(2+)]i. The kinetic parameters of transient changes in Zn(2+) and Ca(2+) under electrical stimulation and the spatiotemporal properties of Zn(2+) and Ca(2+) sparks in resting cells are found to be normal in the treated diabetic group. Biochemical analysis demonstrated that the NAC treatment also antagonized hyperphosphorylation of cardiac ryanodine receptors (RyR2) and significantly restored depleted protein levels of both RyR2 and calstabin2. Incubation of cardiomyocytes with 10 µM ZnCl2 exerted hyperphosphorylation in RyR2 as well as higher phosphorphorylations in both PKA and CaMKII in a concentration-dependent manner, similar to hyperglycemia. Our present data also showed that a subcellular oxidative stress marker, NF-κB, can be activated if the cells are exposed directly to Zn(2+). We thus for the first time report that an enhancement of antioxidant defence in diabetics via directly targeting heart seems to prevent diastolic dysfunction due to modulation of RyR2 macromolecular-complex thereby leading to normalized [Ca(2+)]i and [Zn(2+)]i in cardiomyocytes.