Role of hydroquinone–thiol conjugates in benzene-mediated toxicity
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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