Role of hydroquinone-thiol conjugates in benzene-mediated toxicity

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 18h 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.

Figure 1. 1D-Western blotting of protein adducts in bone marrow

A) HQ-GSH adducts and B) 4-HNE adducts in rat bone marrow following co-administration of phenol (1.1 mmol/kg) and hydroquinone (0.9 mmol/kg). Lane 1,2 and 3,4 are phenol/HQ treated female and male SD rats respectively; STD, ladder; C, vehicle treated control male rat.

Figure 2. The effect of 2,3,5-GS-HQ on γ-GT activity in bone marrow (left) and HL-60 cell (right) lysates

Bone marrow was flushed from the femurs of male Sprague-Dawley rats with 1 ml of Tris/KCl (20 mM/1.15%) containing 1% Triton X-100. The marrow was homogenized, incubated on ice for 30 min, and centrifuged at 800 X g for 10 min. The supernatant was used as the enzyme source. (●) 2,3,5-GS-HQ; (○), Acivicin.

Figure 3. Effect of HQ-GSH conjugates on rat serum erythropoietin levels

Rats were given daily doses of either (●) saline; (○) 2,3,5-GS-HQ (10 μmol/kg, i.v.) or; (□) 2,6-GS-HQ (50 μmol/kg, i.v.). Each point represents the mean ± SEM (n=5). One-way ANOVA; SNK.