| Benzene is an environmental contaminant which produces serious hematological disorders, including aplastic anemia and acute myelogenous leukemia, but must be metabolized to produce its toxic effects. Because thiol conjugation to a variety of polyphenols results in metabolites with increased biological reactivity, we speculated that glutathione (GSH) conjugation to hydroquinone (HQ), a primary metabolite of benzene, might generate hematotoxic metabolites. Male Sprague-Dawley rats and DBA/2 mice were exposed to benzene or a combination of its two major metabolites, HQ and phenol. Several hydroquinone-glutathione (HQ-GSH) con-jugates were subsequently detected in bone marrow, including 2-(glutathion-S-yl)hydroquinone, 2,6-bis-(glutathion- S-yl)hydroquinone, and 2,3,5-tris-(glutathion- S-yl)hydroquinone (TGHQ). 2-(Cystein-S-ylglycine)hydroquinone, 2-(cystein-S-yl)hydroquinone, and 2-(N-acetylcystein- S-yl)hydroquinone were also detected, and in vitro studies confirmed the ability of bone marrow to acetylate 2-(cystein- S-yl)hydroquinone and deacetylate 2-(N-acetylcystein- S-yl)hydroquinone, indicating the presence of a functional mercapturic acid pathway in bone marrow. We subsequently administered either 2,6- bis-(glutathion-S-yl)hydroquinone or TGHQ to rats and observed significant bone marrow toxicity at very low doses.; To determine the mechanism(s) of toxicity, we utilized a human promyelocytic (HL-60) cell line. HL-60 cells represent a good surrogate for CD34 + bone marrow cells, which are proposed targets of benzene in humans. HL-60 cells were found to produce HQ-GSH conjugates when treated with HQ; however, no cysteine conjugate N-acetyltransferase activity was detected. Therefore, immature bone marrow cells may be particularly susceptible to HQ-GSH conjugates, because of their inability to detoxify and eliminate reactive cysteine conjugates. TGHQ, the most potent metabolite in rats, readily induced apoptosis in HL-60 cells. The induction of apoptosis appeared to involve the inhibition of gamma-glutamyl transpeptidase, an enzyme responsible for the bioactivation of HQ-GSH conjugates and the recycling of effluxed GSH. Thus, inhibition of gamma-glutamyl transpeptidase by TGHQ resulted in a depletion in intracellular GSH levels. GSH is a physiological inhibitor of neutral, magnesium-dependent sphingomyelinases. Consequently, TGHQ also causes sphingomyelin turnover in HL-60 cells, leading to a significant increase in intracellular ceramide. Ceramide, a lipid second-messenger, mediates a variety of biological effects, including generation of ROS, activation of the transcription factor NF-kappaB, and activation of the proapoptotic cysteine protease, caspase-3. Similarly, TGHQ-mediated apoptosis was completely inhibited by catalase and partially blocked by various caspase inhibitors. Increased NF-kappaB-DNA binding activity was also observed, and inhibition of this transcription factor resulted in increased apoptosis. In summary, these data suggest that HQ-GSH conjugates induce apoptosis in immature bone marrow cells via the ceramide signaling pathway and that this process may initiate benzene-induced bone marrow suppression. |
