Benzene-derived quinol-thioethers induce apoptosis in hematopoietic tissue via a unique ceramide signaling pathway.

摘要

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.