Myeloperoxidase-catalyzed redox-cycling of phenol promotes lipid peroxidation and thiol oxidation in HL-60 cells

Abstract

Various types of cancer occur in peroxidase-rich target tissues of animals exposed to aryl alcohols and amines. Unlike biotransformation by cytochrome P450 enzymes, peroxidases activate most substrates by one-electron oxidation via radical intermediates. This work analyzed the peroxidase-dependent formation of phenoxyl radicals in HL-60 cells and its contribution to cytotoxicity and genotoxicity. The results showed that myeloperoxidase-catalyzed redox cycling of phenol in HL-60 cells led to intracellular formation of glutathionyl radicals detected as GS-DMPO nitrone. Formation of thiyl radicals was accompanied by rapid oxidation of glutathione and protein-thiols. Analysis of protein sulfhydryls by SDS-PAGE revealed a significant oxidation of protein SH-groups in HL-60 cells incubated in the presence of phenol/H₂O₂ that was inhibited by cyanide and azide. Additionally, cyanide- and azide-sensitive generation of EPR-detectable ascorbate radicals was observed during incubation of HL-60 cell homogenates in the presence of ascorbate and H₂O₂. Oxidation of thiols required addition of H₂O₂ and was inhibited by pretreatment of cells with the inhibitor of heme synthesis, succinylacetone. Radical-driven oxidation of thiols was accompanied by a trend toward increased content of 8-oxo-7,8-dihydro-2′-deoxyguanosine in the DNA of HL-60 cells. Membrane phospholipids were also sensitive to radical-driven oxidation as evidenced by a sensitive fluorescence HPLC-assay based on metabolic labeling of phospholipids with oxidation-sensitive cis-parinaric acid. Phenol enhanced H₂O₂-dependent oxidation of all classes of phospholipids including cardiolipin, but did not oxidize parinaric acid–labeled lipids without addition of H₂O₂. Induction of a significant hypodiploid cell population, an indication of apoptosis, was detected after exposure to H₂O₂ and was slightly but consistently and significantly higher after exposure to H₂O₂/phenol. The clonogenicity of HL-60 cells decreased to the same extent after exposure to H₂O₂ or H₂O₂/phenol. Treatment of HL-60 cells with either H₂O₂ or H₂O₂/phenol at concentrations adequate for lipid peroxidation did not cause a detectable increase in chromosomal breaks. Detection of thiyl radicals as well as rapid oxidation of thiols and phospholipids in viable HL-60 cells provide strong evidence for redox cycling of phenol in this bone marrow-derived cell line.

Introduction

Phenols are good substrates for peroxidases [1], [2]. Peroxidase-catalyzed oxidation of phenolic compounds usually proceeds by phenoxyl radical intermediates [3], [4]. Some phenoxyl radicals can be reduced by GSH (and other reductants) with concomitant activation of oxygen to superoxide (redox-cycling phenols) [5], [6]. This sequence of reactions has been proposed to contribute to the occurrence of cancer in peroxidase-rich tissues [7]. The cellular events by which redox-cycling aryl alcohols and amines cause cytotoxicity and genotoxicity are at present unknown, but increasing evidence suggests that cellular response to stress (e.g., activation of stress-inducible genes) can determine whether the cells survive, transform, or die [8], [9]. The contribution of oxidative stress to the process of carcinogenesis has been well documented [10], [11]. The contribution of redox-cycling to oxidative insult and carcinogenic transformations triggered by phenols and aryl amines is an important unsolved problem.

Phenol is a typical example of the redox-cycling aryl alcohols [12]; it is also one of the major metabolites accumulating in bone marrow during exposure to benzene [13]. Leukemias associated with benzene exposure [14], or secondary leukemias following chemotherapy with the phenolic antitumor drug, etoposide [15] exemplify the unexplored connection between redox-cycling of phenols, cellular response to oxidative stress, and cancer.

The reactions of phenoxyl radicals in the presence of physiologic reductants under aerobic conditions were described in several studies using different purified peroxidases [16], [17], [18], [19]. In spite of this detailed information, the evidence for redox-cycling of phenols in situ is limited [20]. This could be due to (i) difficulty of detection of the reaction intermediates, phenoxyl radicals, in aqueous solutions at room temperature [21], (ii) absence of oxidation products and covalent adducts (phenoxyl radical is continuously reduced back to the starting material) [20], or (iii) cellular repair mechanisms masking the consequences of redox-cycling (e.g., repair of 8-OHdG in the DNA of cells or excision of lipid hydroperoxides by phospholipases) [22]. Two novel biochemical markers of redox-cycling in cells were used in the present study. Thiyl radicals, trapped with DMPO, were detected by HPLC as the EPR-silent GS-DMPO nitrone [23]. Oxidation of all major classes of phospholipids was quantitatively monitored by bleaching of a fluorescent label, cis-parinaric acid (PnA), that was metabolically incorporated into phospholipids of cell membranes [24], [25]. These sensitive markers of radical-driven oxidations were complemented with measurements of thiol oxidation and measurements of 8-OHdG in the DNA. The cellular response to redox-cycling was investigated by measurement of cell viability, colony-forming ability, induction of apoptosis, and, as a measure of gross genotoxicity, measurement of chromosomal breaks in HL-60 cells.