Oxidative stress mediates toxicity of pyridoxal isonicotinoyl hydrazone analogs

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Abstract

Pyridoxal isonicotinoyl hydrazone (PIH) and many of its analogs are effective iron chelators in vivo and in vitro, and are of interest for the treatment of secondary iron overload. Because previous work has implicated the Fe3+–chelator complexes as a determinant of toxicity, the role of iron-based oxidative stress in the toxicity of PIH analogs was assessed. The Fe3+ complexes of PIH analogs were reduced by K562 cells and the physiological reductant, ascorbate. Depletion of the antioxidant, glutathione, sensitized Jurkat T lymphocytes to the toxicity of PIH analogs and their Fe3+ complexes, and toxicity of the chelators increased with oxygen tension. Fe3+ complexes of pyridoxal benzoyl hydrazone (PBH) and salicyloyl isonicotinoyl hydrazone (SIH) caused lipid peroxidation and toxicity in K562 cells loaded with eicosapentenoic acid (EPA), a readily oxidized fatty acid, whereas Fe(PIH)2 did not. The lipophilic antioxidant, vitamin E, completely prevented both the toxicity and lipid peroxidation caused by Fe(PBH)2 in EPA-loaded cells, indicating a causal relationship between oxidative stress and toxicity. PBH also caused concomitant lipid peroxidation and toxicity in EPA-loaded cells, both of which were reversed as its concentration increased. In contrast, PIH was inactive, while SIH was equally toxic toward control and EPA-loaded cells, without causing lipid peroxidation, indicating a much smaller contribution of oxidative stress to the mechanism of toxicity of these analogs. In summary, PIH analogs and their Fe3+ complexes are redox active in the intracellular environment. The contribution of oxidative stress to the overall mechanism of toxicity varies across the series.

Section snippets

PIH analogs and their Fe3+ complexes

PIH analogs were synthesized by the condensation of the corresponding aldehydes and hydrazides as described [16]. Fe complexes were formed by the addition of FeCl3 (Fisher, Fair Lawn, NJ), dissolved in sodium citrate, and chelator, dissolved in NaOH, in a 1:2 molar ratio, and incubation at room temperature for 1 h following neutralization [10].

Toxicity

Jurkat T lymphocytes and K562 cells were cultured in phenol-red-free RPMI-1640 (Cellgro, Herndon, VA) supplemented with 10% FBS and 2 mM glutamine (Gibco,

Toxicity of PIH analogs and their Fe3+ complexes; effect of GSH depletion

The toxicity of PIH analogs, the structures of which are shown in Fig. 1, and their Fe3+ complexes toward Jurkat T lymphocytes was determined after 72 h incubation (Table 1). The structure–activity relationships describing the toxicity of PIH analogs in Jurkat cells were similar to those in K562 cells [10] and SK-N-MC cells [22], suggesting a common mechanism of action of these iron chelators among different cell types. Toxicity increased with the lipophilicity of the analogs, as has been

Redox cycling by Fe complexes of PIH analogs

K562 cells reduce the Fe complexes of PIH analogs (Table 3), as does the ubiquitous cellular reductant, ascorbate. In contrast, cyclic voltammetry of Fe(SBH)2[33] and Fe(NIH)2[34] demonstrated the absence of a reversible redox potential in the biologically accessible range; from these data, it was concluded that reduction of these complexes by cells is not possible. It may be, however, that these experiments were confounded by the relatively slow kinetics of ligand exchange characteristic of

Acknowledgements

This work was supported by grants from the Canadian Institutes for Health Research (P.P.) and University of Linkoping Grant #83081030 (J.N.), and postdoctoral fellowships from the National Cooley’s Anemia Foundation and the Thalassemia Foundation of Canada (J.L.B.).

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    Present address: Heart Foundation Research Center, School of Health Sciences, Griffith University, Southport, Queensland, Australia.

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