Peroxidase-catalyzed pro- versus antioxidant effects of 4 hydroxytamoxifen: Enzyme specificity and biochemical sequelae

Some studies have shown the potential relevance of the oxidation products o f 4-hydroxytamoxifen (4OHTAM) in carcinogenesis. Other studies show 4OHTAM has antioxidant properties. We characterized the one-electron oxidative act ivation reactions of 4OHTAM and three other phenolics, 3-hydroxytamoxifen ( 3OHTAM), 1-(4-hydroxyphenyl)-1,2-diphenylethene, and phenol (PhOH), catalyz ed 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 t he enzymes were directly compared using cis-parinaric acid (PnA)-loaded hum an serum albumin. All phenolics were substrates for the enzymes, but MPx on ly weakly activated 4OHTAM to its phenoxyl radical. In HL60 cells loaded me tabolically 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 phosphat idylethanolamine, phosphatidylserine, and phosphatidylcholine against both H2O2-induced and normal metabolic oxidation. This suggested 4OHTAM is a poo r substrate for intracellular MPx. In rat aorta smooth muscle cells loaded with PnA, 4OHTAM also protected against AMVN-induced peroxidation of these three phospholipids and sphingomyelin, whereas 3OHTAM did not. Spin trappin g 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 th e nitrone adduct under cell-free conditions when activated with HRP. The da ta show that the substrate specificity of a given (myelo)peroxidase determi nes if a phenolic exerts pro(through generation of reactive phenoxyl radica ls) or antioxidant (through radical scavenging) properties in intracellular environments.