Dithiothreitol inhibits the acceleration of DNA strand breaks by human serum albumin in a metal-catalyzed oxidation and γ-irradiation system

Intact or Cys-34 modified human serum albumin (HAS) was significantly damaged and degraded in the metal-catalyzed oxidation (MCO) system containing dithiothreitol (DTT) as an electron donor and, while it lasted, this protein might have prohibited the •OH or H2O2 from attacking DNA. However, in the glutathione (GSH) and ascorbate (non-thiol) MCO system, HSA was not sacrificially degraded and accelerated rather than prevented DNA strand breaks. In contrast, human ceruloplasmin was not sacrificially degraded but significantly prevented DNA strand breaks in both GSH and DTT MCO system. In γ-irradiation system, only DTT attenuated the acceleration of DNA strand breaks by HSA. This protein did not remove more H2O2 in the presence of reduced GSH (thiol-linked peroxidase) than in the absence of GSH. Therefore it seems that in an MCO system, the antioxidant activity of HSA is dependent on the effectiveness of reducing equivalents to induce the exposure of a functional group scavenging •OH or H2O2 by reduction of disulfide bonds in HSA. In the presence of DTT, disulfide bonds in HSA were completely reduced to cysteinyl residues but not significantly converted into cysteines by ascorbate or GSH. On the other hand, human ceruloplasmin has a resistance to reduction. In conclusion, the antioxidant activity of HSA in the DTT MCO system is thought not to be due to peroxidase function by thiol specificity, but due to sacrificial antioxidant activity by the functional groups, such as hydrophobic compounds exposed to the protein surface by DTT. INTRODUCTION ROS and their derivatives have been thought to play a significant role in the pathogenesis of human diseases such as rheumatoid arthritis, atherosclerosis and cancer (1-2). To circumvent the damage caused by the ROS, multiple defense enzyme systems, collectively called antioxidant proteins, are present not only in intracellular fluids but also in extracellular fluids (3-5). Human blood plasma is more sensitive to ROS, which are generated from granulocytes and macrophages during phagocytosis and the reactions of oxygen with a variety of metabolic products derived from cellular metabolism or γ-irradiation (6). Therefore, proteins that are suspected to have antioxidant properties have been identified in extracellular fluids (7-9). Thiol compounds, including tripeptide GSH, are important antioxidant members since they have been shown to scavenge ROS and other oxygen derivatives by enzymatic, as well as non-enzymatic, mechanisms (10). Thiol-linked peroxidase is a class of enzymes exerting peroxidase activity in support of thiol-reducing equivalents. In human blood plasma GSH peroxidase containing selenium has been identified. However, it decomposes hydrogen peroxide (H2O2) when used with GSH in high non-physiological concentration (5 mM) (11). Therefore, the extracellular thioredoxin or glutaredoxin have been shown to be efficient electron donors for selenium-dependent peroxidase rather than GSH (12). Recently it has been proposed that human ceruloplasmin plays a significant role in H2O2-removing peroxidase activity in the presence of DTT or reduced GSH (13, 14). HSA was speculated to be a “sacrificial” antioxidant in blood plasma and extracellular fluid (15, 16). In contrast, a recent study has suggested that HSA functions as an extracelluar enzymatic antioxidant, and should be classified as a “GSH (thiol)-dependent peroxidase”, not as a “sacrificial” antioxidant (17). It has been shown that an MCO system, which is composed of trace amount of iron, O2 and electron donors, induces strand breaks in plasmid and mammalian DNA as well as the inactivation of several enzymes (18, 19). The aim of the present study is to investigate the antioxidant role of HSA and the effect of DTT on the DNA strand breaks by •OH or H2O2 produced in an MCO and γ-irradiation system. We also provide the first demonstration that the antioxidant property of HSA is different from that of ceruloplasmin, which has been suspected of GSH (thiol)-linked peroxidase, in the MCO system containing thiol compounds. MATERIALS AND METHODS Materials All reagents were of analytical grade. Ammonium sulfate, agarose, GSH, β-mercaptoethanol, DTT, trichloroacetic acid, 5,5’-dithiobis-(2-nitrobenzoic acid), ascorbic acid, ovalbumin, cysteine and HEPES were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Human ceruloplasmin was obtained from Calbiochem. DEAE-Sepharose, pUC and φX174 plasmid DNA were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Cibacron Blue F3-GA-sepharose was supplied by Bio-Rad (Richmond, CA, U.S.A.) and ultrafiltration membrane was purchased from Amicon Co. (Beverly, MA, U.S.A.). KSCN, NaCl, FeCl3 and