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Redox inactivation of glutathione reductase involves metal cations, since chelators protected against NADPH-inactivation, 3 microM EDTA or 10 microM DETAPAC yielding full protection. Ag+, Zn2+ and Cd2+ potentiated the redox inactivation promoted by NADPH alone, while Cr3+, Fe2+, Fe3+, Cu+, and Cu2+ protected the enzyme. The Zn2+ and Cd2+ effect was time-dependent, unlike conventional inhibition. Glutathione reductase interconversion did not require dioxygen, excluding participation of active oxygen species produced by NADPH and metal cations. One Zn2+ ion was required per enzyme subunit to yield full NADPH-inactivation, the enzyme being reactivated by EDTA. Redox inactivation of glutathione reductase could arise from the blocking of the dithiol formed at the active site of the reduced enzyme by metal cations, like Zn2+ or Cd2+. The glutathione reductase activity of yeast cell-free extracts was rapidly inactivated by low NADPH or moderate NADH concentrations; NADP+ also promoted rapid inactivation in fresh extracts, probably after reduction to NADPH. Full inactivation was obtained in cell-free extracts incubated with glucose-6-phosphate or 6-phosphogluconate; the inactivating efficiency of several oxidizable substrates was directly proportional to the specific activities of the corresponding dehydrogenases, confirming that redox inactivation derives from NADPH formed in vitro.

The toxicity of iron (II) and iron (III) chlorides was studied at different biochemical and cellular levels, including antioxidative and metabolic enzymes and two general indicators of cytotoxicity in Vero monkey kidney cells after 24-h exposure. Iron (II) was fourfold more toxic than Fe (III) in cell proliferation, with EC50 of 5.5 and 22 mM, respectively. Metabolic markers were far more sensitive than cytotoxicity assays at these concentrations. At the highest concentrations of toxicant tested [10 mM Fe(II) and 50 mM Fe(III)], both species produced nearly total inhibition of the relative uptake of neutral red (RNRU) and phosphofructokinase activity (PFK), and stimulated intracellular specific lactate dehydrogenase activity (LDH). Succinate dehydrogenase (SDH) and hexosaminidase (HEX) activities were reduced in dose-dependent manner, as was the antioxidative enzyme glucose-6-phosphate dehydrogenase (G-6-PDH) with both forms of iron. Glutathione reductase (GOR) and glutathione-S-transferase (GST) activities were stimulated by Fe (II) but were inhibited by the higher Fe (III) concentrations. In conclusion, the experimental model may be useful for the study of different metabolic effects induced by the two oxidation states of iron.

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