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Antioxidant activity is believed to be an important intracellular function of metallothioneins (MT), yet the specific mechanisms of their antioxidant action are not known. Under conditions when cells are challenged with elevated concentrations of free copper as a result of metabolic disturbances or environmental and occupational exposures, MTs may be ideally suited for antioxidant function as effective copper chelators. In the study presented here, we tested this hypothesis using a recently established model of copper nitrilotriacetate-induced oxidative stress in HL-60 cells. Since copper-induced oxidative stress triggers apoptosis, we further investigated antiapoptotic function of MTs in HL-60 cells. Using a Sephadex G-75 chromatographic partial purification of MTs from cell homogenates with subsequent immuno-dot-blot assay, we showed that zinc pretreatment yielded a pronounced induction of MTs in HL-60 cells. We report that zinc-induced MTs were able to (i) completely bind intracellular copper, (ii) completely quench redox-cycling activity of copper, (iii) significantly inhibit copper-dependent oxidative stress in membrane phospholipids, and (iv) prevent copper-dependent apoptosis and its characteristic biochemical features (cytochrome c release from mitochondria into cytosol, caspase-3 activation, and externalization of phosphatidylserine in plasma membranes). In separate experiments, we used lung fibroblasts derived from MT1, MT2 knockout mice (MT^-/-) and MT wild-type (MT^+/+) mice. ZnCl2 pretreatment resulted in a more than 10-fold induction of MTs in MT^+/+ cells, whereas the MT content in MT^-/- cells remained low, at levels ≈100-fold lower than in their MT wild-type counterparts. MT^-/- cells were very sensitive to Cu-NTA and, most importantly, showed no response to ZnCl2 pretreatment. In contrast, MT^+/+ cells were relatively more resistant to Cu-NTA, and this resistance was remarkably enhanced by ZnCl2 pretreatment. Combined, our results demonstrate that metallothioneins function as effective antioxidants and an antiapoptotic mechanism in copper-challenged HL-60 cells.

Etoposide (VP-16) is extensively used to treat cancer, yet its efficacy is calamitously associated with an increased risk of secondary acute myelogenous leukemia. The mechanisms for the extremely high susceptibility of myeloid stem cells to the leukemogenic effects of etoposide have not been elucidated. We propose a mechanism to account for the etoposide-induced secondary acute myelogenous leukemia and nutritional strategies to prevent this complication of etoposide therapy. We hypothesize that etoposide phenoxyl radicals (etoposide-O(.)) formed from etoposide by myeloperoxidase are responsible for its genotoxic effects in bone marrow progenitor cells, which contain constitutively high myeloperoxidase activity. Here, we used purified human myeloperoxidase, as well as human leukemia HL60 cells with high myeloperoxidase activity and provide evidence of the following. 1) Etoposide undergoes one-electron oxidation to etoposide-O(.) catalyzed by both purified myeloperoxidase and myeloperoxidase activity in HL60 cells; formation of etoposide-O(.)radicals is completely blocked by myeloperoxidase inhibitors, cyanide and azide. 2) Intracellular reductants, GSH and protein sulfhydryls (but not phospholipids), are involved in myeloperoxidase-catalyzed etoposide redox-cycling that oxidizes endogenous thiols; pretreatment of HL60 cells with a maleimide thiol reagent, ThioGlo1, prevents redox-cycling of etoposide-O(.) radicals and permits their direct electron paramagnetic resonance detection in cell homogenates. VP-16 redox-cycling by purified myeloperoxidase (in the presence of GSH) or by myeloperoxidase activity in HL60 cells is accompanied by generation of thiyl radicals, GS(.), determined by HPLC assay of 5, 5-dimethyl-1-pyrroline glytathionyl N-oxide glytathionyl nitrone adducts. 3) Ascorbate directly reduces etoposide-O(.), thus competitively inhibiting etoposide-O(.)-induced thiol oxidation. Ascorbate also diminishes etoposide-induced topo II-DNA complex formation in myeloperoxidase-rich HL60 cells (but not in HL60 cells with myeloperoxidase activity depleted by pretreatment with succinyl acetone). 4) A vitamin E homolog, 2,2,5,7, 8-pentamethyl-6-hydroxychromane, a hindered phenolic compound whose phenoxyl radicals do not oxidize endogenous thiols, effectively competes with etoposide as a substrate for myeloperoxidase, thus preventing etoposide-O(.)-induced redox-cycling. We conclude that nutritional antioxidant strategies can be targeted at minimizing etoposide conversion to etoposide-O(.), thus minimizing the genotoxic effects of the radicals in bone marrow myelogenous progenitor cells, i.e., chemoprevention of etoposide-induced acute myelogenous leukemia.

Some studies have shown the potential relevance of the oxidation products of 4-hydroxytamoxifen (4OHTAM) in carcinogenesis. Other studies show 4OHTAM has antioxidant properties. We characterized the one-electron oxidative activation reactions of 4OHTAM and three other phenolics, 3-hydroxytamoxifen (3OHTAM), 1-(4-hydroxyphenyl)-1,2-diphenylethene, and phenol (PhOH), catalyzed by myeloperoxidase (MPx), horseradish peroxidase (HRP), lactoperoxidase, mushroom tyrosinase, and nonenzymatic initiators in vitro under a variety of conditions and in cells. Differences in activation of the phenolics by the enzymes were directly compared using cis-parinaric acid (PnA)-loaded human serum albumin. All phenolics were substrates for the enzymes, but MPx only weakly activated 4OHTAM to its phenoxyl radical. In HL60 cells loaded metabolically with PnA so that effects on phospholipids could be monitored by HPLC with fluorescence detection, PhOH plus H2O2 caused massive oxidation across all phospholipid classes. 4OHTAM dose-dependently protected phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine against both H2O2-induced and normal metabolic oxidation. This suggested 4OHTAM is a poor substrate for intracellular MPx. In rat aorta smooth muscle cells loaded with PnA, 4OHTAM also protected against AMVN-induced peroxidation of those three phospholipids and sphingomyelin, whereas 3OHTAM did not. Spin trapping of glutathionyl radicals (GS^•) with DMPO and quantifying the ESR-silent nitrone form of the GS−DMPO adduct by HPLC showed that neither 3OHTAM plus H2O2 nor 4OHTAM plus H2O2 caused a significant level of GSH oxidation with isolated MPx, nor did the latter in HL60 cells, whereas PhOH plus H2O2 was a potent source of GS^• in both systems. Both 4OHTAM and 3OHTAM formed the nitrone adduct under cell-free conditions when activated with HRP. The data show that the substrate specificity of a given (myelo)peroxidase determines if a phenolic exerts pro- (through generation of reactive phenoxyl radicals) or antioxidant (through radical scavenging) properties in intracellular environments.