Role of hydroquinone–thiol conjugates in benzene-mediated toxicity

https://doi.org/10.1016/j.cbi.2009.12.016Get rights and content

Abstract

Hydroquinone (HQ) is a metabolite of benzene, and in combination with phenol (PHE), reproduces benzene myelotoxicity. HQ readily oxidizes to 1,4-benzoquinone (1,4-BQ) followed by the reductive addition of glutathione (GSH). Subsequent cycles of oxidation and GSH addition give rise to a variety of mono-, and multi-GSH substituted conjugates. Following administration of PHE/HQ (1.1 mmol/kg/0.9 mmol/kg, ip) to male Sprague–Dawley (SD) rats, 2-(glutathion-S-yl)HQ [GS-HQ], 2,5-bis-(glutathion-S-yl)HQ [2,5-GS-HQ], 2,6-bis-(glutathion-S-yl)HQ [2,6-GS-HQ], and 2,3,5-tris-(glutathion-S-yl)HQ [2,3,5-GS-HQ] were all identified in bone marrow. 2-(Cystein-S-ylglycine)HQ [2-(CysGly)HQ], 2-(cystein-S-yl)HQ [2-(Cys)HQ], and 2-(N-acetylcystein-S-yl)HQ [2-(NACys)HQ] were also found in the bone marrow of PHE/HQ and benzene treated rats and mice, indicating the presence of an active mercapturic acid pathway within bone marrow. Moreover, 2,6-GS-HQ and 2,3,5-GS-HQ were hematotoxic when administered to rats. All of the HQ–GSH conjugates retain the ability to redox cycle and generate reactive oxygen species (ROS), and to arylate target proteins. Recent in vitro and in vivo studies in our laboratory revealed lysine and arginine residues as primary targets of 1,4-BQ, GS-HQ and 2-(NACys)HQ adduction. In contrast 1,4-BQ-adduction of cysteine residues may be a transient interaction, where physiological conditions dictate adduct stability. The generation of ROS and alkylation of proteins may both contribute to benzene-mediated myelotoxicity, and the two processes may be inter-dependent. However, the precise molecular mechanism by which benzene and HQ–GSH conjugates induce hematotoxicity remains to be determined. Within 18 h of administration of PHE/HQ to SD rats a significant decrease in blood lymphocyte count was observed. At this early time point, erythrocyte counts and hemoglobin concentrations remained within the normal range. Concomitant with the decrease in lymphocyte count, western blot analysis of bone marrow lysate, using HQ–GSH and 4-hydroxy-2-nonenal (4HNE) specific antibodies, revealed the presence of HQ–GSH- and 4HNE-derived protein adducts. Identification of these adducts is required before the functional significance of such protein modifications can be determined.

Introduction

Benzene, a major industrial chemical and environmental pollutant, causes a variety of hematological disorders in man, including aplastic anemia, myelodysplastic syndrome, and acute myelogenous leukemia. While benzene must be metabolized to yield its hematotoxic and leukemogenic effects, no single metabolite of benzene reproduces these effects in vivo. Co-administration of PHE and HQ, however, does lead to myelotoxicity in rodents [1]. A pharmacokinetic interaction between these two metabolites results in increased concentrations of both metabolites in bone marrow [2]. Peroxidase and/or phenoxy-radical mediated oxidation of HQ then theoretically initiates redox cycling and formation of the reactive electrophile, 1,4-BQ, which is considered to be one of the ultimate hematotoxic metabolites of benzene [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. 1,4-BQ is an electrophile, and covalent interactions of quinones with nucleophilic sites within cellular macromolecules may contribute to the toxic effects of benzene [7], [14], [15], [16]. Indeed, the combined treatment of PHE and [14C]-HQ increases myelotoxicity with concomitant increases in covalently bound radiolabel in blood and bone marrow [10]. Moreover, elevated levels of benzene oxide and HQ-derived (1,4-BQ?) adducts of hemoglobin and albumin have been observed in workers subjected to benzene exposure [17], [18].

Focusing on cysteine-targeted protein adducts, McDonald et al. [19] reported that 1,4-BQ protein binding was favored over benzene oxide in mouse bone marrow. Although modification on selective target proteins occurs in bone marrow of mice following treatment with [14C]-benzene [20], the exact nature of the adducted metabolite(s) and/or the specific site of adduction on target proteins are not known. Of particular relevance to the ability of benzene to induce aneuploidy, and other forms of chromosomal aberrations, histones were identified as potential targets of unknown reactive benzene metabolites [20]. 1,4-BQ readily conjugates with glutathione (GSH) to give 2-GS-HQ, 2,3-GS-HQ, 2,5-GS-HQ, 2,6-GS-HQ and 2,3,5-GS-HQ [21]. Moreover, HQ–GSH conjugates are present in the bone marrow of rats and mice following co-administration of PHE/HQ [22] and metabolized to more reactive cysteinylgylcine and cysteine conjugates via the mercapturic acid pathway in bone marrow. Because HQ–thioether metabolites have an enhanced capacity to both redox cycle [Monks et al. (this issue)] and arylate tissue macromolecules [23], [24], we suggest that they play an important role in benzene-mediated toxicity via a mechanism involving the production of ROS and/or macromolecular arylation. Interestingly, lysine residues appear to be a preferred target of quinone–thiother adduction [24], [25]. ROS produced as a result of HQ–thioether redox cycling are also capable of oxidatively modifying both proteins and DNA thereby producing toxicity. Herein we report the presence of HQ–thioether and 4HNE-derived protein adducts following in vivo administration of PHE/HQ to rats. These two inter-dependent pathways of protein modification may contribute to benzene induced myleotoxicity.

Section snippets

Materials

HQ and PHE were purchased from Sigma–Aldrich (St. Louis, MO). Cell Lysis Buffer (10×) was purchased from Cell Signaling Technology (Danvers, MA). Complete Protease Inhibitor Cocktail Tablets were purchased from Roche (Madison, WI). Antibody sources were as follows: rabbit anti-2-Br-6-(N-acetylcystein-S-yl)hydroquinone (anti-2-BrHQ-NAC) in-house [26]; anti-4HNE antibody was a generous gift from Dr. Dennis R. Petersen (University of Colorado Health Sciences Center) produced and characterized as

Effect of blood counts after phenol/hydroquinone administration

Complete blood counts of rats 18 h following co-administration of PHE/HQ (1.1/0.9 mmol/kg, ip) to rats revealed significant reduction (58–89%) of lymphocyte counts when compared to control values (Table 1). All other leukocyte and erythrocyte counts were within their normal ranges following treatment.

1D western detection of HQ–GSH/4HNE-protein adducts in bone marrow

It should be noted that immediately following co-administration of phenol/hydroquinone, a transient (15–20 min) neurological side-effect is observed. Interestingly the intensity of this side-effect

Discussion

HQ–thioethers are present in the bone marrow of rats following co-administration of PHE/HQ, the majority of which appear to be generated in situ and further metabolized via the mercapturic acid pathway [22]. This pathway is important in modulating the reactivity of HQ–GSH conjugate. Based on the (re)activity of HQ–GSH conjugates, we speculated that some of the hematotoxic effects attributed to HQ (or 1,4-BQ) may, in fact, be mediated by their thiol conjugates. Indeed, as shown by Monks et al

Conflict of interest

None.

Acknowledgements

This work was supported by RO1 GM070890 (SSL). The authors also acknowledge the support of the P30 ES006694 Southwest Environmental Health Sciences Center, in particular the Arizona Proteomics Consortium (APC) and Dr. George Tsaprailis, Director of the APC. The generous gift of anti-4HNE antibody from Dr. Dennis R. Petersen at the University of Colorado Health Sciences Center is greatly appreciated.

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