Protective effect of zinc-N-acetylcysteine on the rat kidney during cold storage.

Cold storage of kidneys before transplantation is problematic because of the limited survival time of the allografts. In this study, zinc-N-acetylcysteine (ZnNAC) was shown to be a potent endonuclease inhibitor and antioxidant, and it was tested as a potential additive to a cold storage solution for kidney preservation. Exposure of normal rat kidney NRK-52E cells to ZnNAC resulted in zinc delivery to the cells as determined by TFL-Zn fluorophore and partial protection of the cells against injury by cold storage in University of Wisconsin solution (UWS) as measured by propidium iodide assay. Ex vivo, rat kidneys demonstrated time- and temperature-dependent DNA fragmentation as assessed by TUNEL assay, indicating irreversible cell death. DNA fragmentation was faster in the medulla than in the cortex, and tubules were affected more than glomeruli. Perfusion of rat kidneys with cold ZnNAC solution in UWS significantly inhibited cell death both in the cortex and medulla at concentrations of 0.3-30 mM compared with UWS alone, with a maximum effect at 1-10 mM ZnNAC. Cold storage of the kidney significantly increased quantities of cleaved caspase-3 and endonuclease G (EndoG) in the tissue, which were abolished by 10 mM ZnNAC, indicating its ability to suppress both caspase-dependent and -independent cell death. Therefore, supplementation of UWS with ZnNAC can decrease DNA fragmentation and protect kidney allografts from cell death due to cold storage.

Deoxyribonuclease I is essential for DNA fragmentation induced by gamma radiation in mice.

Gamma radiation is known to induce cell death in several organs. This damage is associated with endonuclease-mediated DNA fragmentation; however, the enzyme that produces the latter and is likely to cause cell death is unknown. To determine whether the most abundant cytotoxic endonuclease DNase I mediates gamma-radiation-induced tissue injury, we used DNase I knockout mice and zinc chelate of 3,5-diisopropylsalicylic acid (Zn-DIPS), which, as we show, has DNase I inhibiting activity in vitro. The study demonstrated for the first time that inactivation or inhibition of DNase I ameliorates radiation injury to the white pulp of spleen, intestine villi and bone marrow as measured using a quantitative TUNEL assay. The spleen and intestine of DNase I knockout mice were additionally protected from radiation by Zn-DIPS, perhaps due to the broad radioprotective effect of the zinc ions. Surprisingly, the main DNase I-producing tissues such as the salivary glands, pancreas and kidney showed no effect of DNase I inactivation. Another unexpected observation was that even without irradiation, DNA fragmentation and cell death were significantly lower in the intestine of DNase I knockout mice than in wild-type mice. This points to the physiological role of DNase I in normal cell death in the intestinal epithelium. In conclusion, our results suggested that DNase I-mediated mechanism of DNA damage and subsequent tissue injury are essential in gamma-radiation-induced cell death in radiosensitive organs.

Thorough study of reactivity of various compound classes toward the Folin-Ciocalteu reagent.

A thorough study was done to test the reactivity of the Folin-Ciocalteu (F-C) reagent toward various compound classes. Over 80 compounds were tested. Compound classes included phenols, thiols, vitamins, amino acids, proteins, nucleotide bases, unsaturated fatty acids, carbohydrates, organic acids, inorganic ions, metal complexes, aldehydes, and ketones. All phenols, proteins, and thiols tested were reactive toward the reagent. Many vitamin derivatives were also reactive, as were the inorganic ions Fe(+2), Mn(2+), I(-), and SO(3)(2-). Other compounds showing reactivity included the nucleotide base guanine and the trioses glyceraldehyde and dihydroxyacetone. Copper complexation enhanced the reactivity of salicylate derivatives toward the reagent, whereas zinc complexation did not. Several amino acids and sugars that were reported to be reactive toward the F-C reagent in earlier studies were found not to be reactive in this study, at least in the concentrations used. Reaction kinetics of each compound with the F-C reagent were also measured. Most compounds tested showed a biphasic kinetic pattern with half-lives under 1 min. Trolox and ascorbic acid displayed a rapid monophasic pattern in which the reaction reached end point within 1 min. In summary, this study has shown that the F-C reagent is significantly reactive toward other compounds besides phenols. As other investigators have suggested, the F-C assay should be seen as a measure of total antioxidant capacity rather than phenolic content. Because phenolics are the most abundant antioxidants in most plants, it gives a rough approximation of total phenolic content in most cases.

Comparative reaction rates of various antioxidants with ABTS radical cation.

The reaction rates of several aminothiol, amidothiol, and phenolic antioxidants with ABTS radical cation were measured. Most compounds had half-lives of less than one minute. However several compounds had considerably longer half-lives. Aminothiol derivatives lacking a free thiol group, such as amifostine and RibCys, displayed longer half-lives. Reaction of these compounds with the ABTS radical cation displayed first order kinetic behavior. Of the phenolic compounds studied, chlorogenic acid and caffeic acid had the longest half-lives. Most phenolics displayed a biphasic kinetic pattern involving fast and slow steps. Some of the aminothiols also displayed this type of behavior. Glutathione disulfide was reactive toward ABTS radical cation and displayed slow kinetics. This suggests that the slow step observed with some of the aminothiols may be due to initial rapid formation of disulfide followed by slow reaction of the disulfide with ABTS radical cation. Some compounds required a considerably longer incubation time to reach end point than the six to ten minute period normally used for this assay. This suggests that, when ABTS is being used as an end point assay, a longer incubation time may be needed to obtain reliable data. When food substances are being tested using this assay, kinetic profiles should first be examined before end points are determined. This paper contains the first published data reporting antioxidant capacities of amino- and amidothiols measured by the ABTS method.