Pyridoxal isonicotinoyl hydrazone (PIH) prevents copper-mediated in vitro free radical formation

Pyridoxal isonicotinoyl hydrazone (PIH) is an iron chelator with antioxidant activity, low toxicity and is useful in the experimental treatment of iron-overload diseases. Previous studies on x-ray diffraction have revealed that PIH also forms a complex with Cu(II). Since the main drug of choice for the treatment of Wilson's disease, d-penicillamine, causes a series of side effects, there is an urgent need for the development of alternative copper chelating agents for clinical use. These chelators must also have antioxidant activity because oxidative stress is associated with brain and liver copper-overload. In this work we tested the ability of PIH to prevent in vitro free radical formation mediated by Cu(II), ascorbate and dissolved O-2. Degradation of 2-deoxyribose mediated by 10 muM Cu(II) and 3 mM ascorbate was fully inhibited by 10 muM PIH (I-50 = 6 muM) or 20 muM d-penicillamine (I-50 = 10 muM). The antioxidant efficiency of PIH remained unchanged with increasing concentrations (from 1 to 15 mM) of the hydroxyl radical detector molecule, 2-deoxyribose, indicating that PIH does not act as a hydroxyl scavenger. On the other hand, the efficiency of PIH (against copper-mediated 2-deoxyribose degradation and ascorbate oxidation) was inversely proportional to the Cu(II) concentration, suggesting a competition between PIH and ascorbate for complexation with Cu(II). An almost full inhibitory effect by PIH was observed when the ratio PIH:copper was 1:1. A similar result was obtained with the measurement of copper plus ascorbate-mediated O-2 uptake. Moreover, spectral studies of the copper and PIH interaction showed a peak at 455 nm and also indicated the formation of a stable Cu(II) complex with PIH with a 1:1 ratio. These data demonstrated that PIH prevents hydroxyl radical formation and oxidative damage to 2-deoxyribose by forming a complex with Cu(II) that is not reactive with ascorbate (first step of the reactions leading to hydroxyl radical formation from Cu(II), ascorbate and O-2) and does not participate in Haber-Weiss reactions.