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Title: Inhibition of human DNA topoisomerase II by hydroquinone and p-benzoquinone, reactive metabolites of benzene

Abstract

Chronic exposure of humans to benzene (BZ) causes acute myeloid leukemia (AML). Both BZ and therapy-related secondary AML are characterized by chromosomal translocations that may occur by inappropriate recombinational events. DNA topoisomerase 11 (topo 11) is an essential sulfhydryl (SH)-dependent endonuclease required for replication, recombination, chromosome segregation, and chromosome structure. Topo 11 cleaves DNA at purine(R)/pyrimidine(Y) repeat sequences that have been shown to be highly recombinogenic in vivo. Certain antineoplastic drugs stabilize topo 11-DNA cleavage complexes at RY repeat sequences, which leads to translocations of the type observed in leukemia. Hydroquinone (HQ) is metabolized to p-benzoquinone (BQ) in a peroxidase-mediated reaction in myeloid progenitor cells. BO interacts with SH groups of SH-dependent enzymes. Consequently, the aims of this research were to determine whether HQ and BO are topo 11 inhibitors. The ability of the compounds to inhibit the activity of topo, 11 was tested using an assay system that depends on the conversion, by homogeneous human topo 11, of catenated kinetoplast DNA into open and/or nicked open circular DNA that can be separated from the catenated DNA by electrophoresis in a 1% agarose-ethidium bromide gel. We provide preliminary data that indicate that both HQ and BO cause a time andmore » concentration (pM)-dependent inhibition of topo 11 activity. 32 refs., 5 figs.« less

Authors:
;  [1]
  1. Thomas Jefferson Univ., Philadelphia, PA (United States)
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
472170
Report Number(s):
CONF-9506288-
Journal ID: EVHPAZ; ISSN 0091-6765; TRN: 97:001626-0022
Resource Type:
Journal Article
Resource Relation:
Journal Name: Environmental Health Perspectives; Journal Volume: 104; Journal Issue: Suppl.6; Conference: Benzene `95: international conference on benzene toxicity, carcinogenesis, and epidemiology, Piscataway, NJ (United States), 17-20 Jun 1995; Other Information: PBD: Dec 1996
Country of Publication:
United States
Language:
English
Subject:
02 PETROLEUM; 55 BIOLOGY AND MEDICINE, BASIC STUDIES; BENZENE; METABOLISM; QUINONES; BIOLOGICAL EFFECTS; ISOMERASES; INHIBITION; OCCUPATIONAL EXPOSURE; EXHAUST GASES; AIR POLLUTION; PETROLEUM REFINERIES; CARCINOGENS; ANTINEOPLASTIC DRUGS; LEUKEMOGENESIS; MYELOID LEUKEMIA; STRAND BREAKS; ENZYME INHIBITORS

Citation Formats

Hutt, A.M., and Kalf, G.F. Inhibition of human DNA topoisomerase II by hydroquinone and p-benzoquinone, reactive metabolites of benzene. United States: N. p., 1996. Web. doi:10.2307/3433173.
Hutt, A.M., & Kalf, G.F. Inhibition of human DNA topoisomerase II by hydroquinone and p-benzoquinone, reactive metabolites of benzene. United States. doi:10.2307/3433173.
Hutt, A.M., and Kalf, G.F. 1996. "Inhibition of human DNA topoisomerase II by hydroquinone and p-benzoquinone, reactive metabolites of benzene". United States. doi:10.2307/3433173.
@article{osti_472170,
title = {Inhibition of human DNA topoisomerase II by hydroquinone and p-benzoquinone, reactive metabolites of benzene},
author = {Hutt, A.M. and Kalf, G.F.},
abstractNote = {Chronic exposure of humans to benzene (BZ) causes acute myeloid leukemia (AML). Both BZ and therapy-related secondary AML are characterized by chromosomal translocations that may occur by inappropriate recombinational events. DNA topoisomerase 11 (topo 11) is an essential sulfhydryl (SH)-dependent endonuclease required for replication, recombination, chromosome segregation, and chromosome structure. Topo 11 cleaves DNA at purine(R)/pyrimidine(Y) repeat sequences that have been shown to be highly recombinogenic in vivo. Certain antineoplastic drugs stabilize topo 11-DNA cleavage complexes at RY repeat sequences, which leads to translocations of the type observed in leukemia. Hydroquinone (HQ) is metabolized to p-benzoquinone (BQ) in a peroxidase-mediated reaction in myeloid progenitor cells. BO interacts with SH groups of SH-dependent enzymes. Consequently, the aims of this research were to determine whether HQ and BO are topo 11 inhibitors. The ability of the compounds to inhibit the activity of topo, 11 was tested using an assay system that depends on the conversion, by homogeneous human topo 11, of catenated kinetoplast DNA into open and/or nicked open circular DNA that can be separated from the catenated DNA by electrophoresis in a 1% agarose-ethidium bromide gel. We provide preliminary data that indicate that both HQ and BO cause a time and concentration (pM)-dependent inhibition of topo 11 activity. 32 refs., 5 figs.},
doi = {10.2307/3433173},
journal = {Environmental Health Perspectives},
number = Suppl.6,
volume = 104,
place = {United States},
year = 1996,
month =
}
  • Benzene is a clastogenic and carcinogenic agent that induces acute myelogenous leukemia in humans and multiple types of tumors in animals. Previous research has indicated that benzene must first be metabolized to one or more bioactive species to exert its myelotoxic and genotoxic effects. To better understand the possible role of individual benzene metabolites in the leukemogenic process, as well as to further investigate inhibition of topoisomerase 11 by benzene metabolites, a series of known and putative benzene metabolites, phenol, 4{prime}4-biphenol, 2,2{prime}-biphenol, hydroquinone, catechol, 1,2,4-benzenetriol, 1,4-benzoquinone, and trans-trans-muconaldehyde were tested for inhibitory effects in vitro on the human topoisomerase 11more » enzyme. With minor modifications of the standard assay conditions, 1,4-benzoquinone and trans-trans-muconaldehyde were shown to be directly inhibitory, whereas all of the phenolic metabolites were shown to inhibit enzymatic activity following bioactivation using a peroxidase activation system. The majority of compounds tested inhibited topoisomerase 11 at concentrations at or below 10 pM. These results confirm and expand upon previous findings from our laboratory and indicate that many of the metabolites of benzene could potentially interfere with topoisomerase 11. Since other inhibitors of topoisomerase 11 have been shown to induce leukemia in humans, inhibition of this enzyme by benzene metabolites may also play a role in the carcinogenic effects of benzene. 48 refs., 4 tabs.« less
  • Chronic exposure of humans to benzene affects hematopoietic stem and progenitor cells and leads to aplastic anemia. The stromal macrophage, a target of benzene toxicity, secretes interieukin-1 (IL-1), which induces the stromal fibroblast to synthesize hematopoietic colony-stimulating factors. In a mouse model, benzene causes an acute marrow hypocellularity that can be prevented by the concomitant administration of IL-1{alpha}. The ability of benzene to interfere with the production and secretion of IL-1{alpha} was tested. Stromal macrophages from benzene-treated mice were capable of the transcription of the IL-1{alpha} gene and the translation of the message but showed an inability to process themore » 34-kDa pre-IL-1{alpha} precursor to the 17-kDa biologically active cytokine. Treatment of normal murine stromal macrophages in culture with hydroquinone (HQ) also showed an inhibition in processing of pre-IL-1{alpha}. Hydroquinone is oxidized by a peroxidase-mediated reaction in the stromal macrophage to p-benzoquinone, which interacts with the sulfhydryl (SH) groups of proteins and was shown to completely inhibit the activity of calpain, the SH-dependent protease that cleaves pre-IL-1{alpha}. In a similar manner, HQ, via peroxidase oxidation to p-benzoquinone, was capable of preventing the IL-1{beta} autocrine stimulation of growth of human B1 myeloid tumor cells by preventing the processing of pre-IL-1{beta} to mature cytokine. Benzoquinone was also shown to completely inhibit the ability of the SH-dependent IL-1{beta} converting enzyme. Thus benzene-induced bone marrow hypocellularity may result from apoptosis of hematopoietic progenitor cells brought about by lack of essential cylokines and deficient IL-1{alpha} production subsequent to the inhibition of calpain by p-benzoquinone and the prevention of pre-IL-1 processing. 34 refs., 8 figs.« less
  • HL-60/AMSA is a human leukemia cell line that is 50-100-fold more resistant than its drug-sensitive HL-60 parent line to the cytotoxic actions of the DNA intercalator amsacrine (m-AMSA). HL-60/AMSA topoisomerase II is also resistant to the inhibitory actions of m-AMSA. HL-60/AMSA cells and topoisomerase II are cross-resistant to anthracycline and ellipticine intercalators but relatively sensitive to the nonintercalating topoisomerase II reactive epipodophyllotoxin etoposide. The authors now demonstrate that HL-60/AMSA and its topoisomerase II are cross-resistant to the DNA intercalators mitoxantrone and amonafide, thus strongly indicating that HL-60/AMSA and its topoisomerase II are resistant to topoisomerase II reactive intercalators but notmore » to nonintercalators. At high concentrations, mitoxantrone and amonafide were also found to inhibit their own, m-AMSA's, and etoposide's abilities to stabilize topoisomerase II-DNA complexes. These results suggest that the cytotoxicity of m-AMSA and etoposide is initiated primarily by the stabilization of the topoisomerase II-DNA complex. Other topoisomerase II reactive drugs may inhibit the enzyme at other steps in the topoisomerization cycle, particularly at elevated concentrations. Under these conditions, these latter drugs may not produce protein-associated DNA cleavage while still inhibiting topoisomerase II function as well as the actions of other topoisomerase II reactive drugs.« less
  • This work describes the use of protein binding to study the tissue doses of two classes of DNA-reactive metabolites resulting from administration of benzene to rats and mice. A desulfurization catalyst (Raney{reg_sign} nickel) formed the basis of a method to measure adducts of benzoquinones (BQ) with cysteinyl residues of proteins. Sulfur-bound adducts were cleaved by Raney{reg_sign} nickel, extracted, derivatized and analyzed by GC-ECD or GC-MS. Reactions of 1,4-BQ with whole blood of humans, rats and mice, in vitro, were undertaken to compare dose-related formation of BQ adducts. Rate constants for reactions of BQ with blood and blood proteins were estimated.more » Binding with hemoglobin (Hb) and bone-marrow proteins was used to compare in vivo reactions of four electrophilic metabolites of benzene, benzene oxide, 1,2-BQ, 1,4-BQ and 4,4{prime}-diphenoquinone, in F344 rats and B6C3F{sub 1} mice. Animals were given a single oral administration of [{sup 13}C{sub 6}] benzene and/or [{sup 14}C]benzene. The proportions of total protein binding were estimated for each reactive metabolite. Dose-related production of adducts of benzene oxide, 1,2-BQ and 1,4-BQ with Hb and bone-marrow proteins was seen in both species. Interestingly, large differences in adduct formation were observed between species and tissues. In the rat, benzene-oxide adducts of Hb predominated in the blood, whereas 1,2-BQ adducts were more prevalent in the marrow. In the mouse, adducts of 1,4-BQ were of greatest abundance with both Hb and bone-marrow proteins. The average blood concentrations of 1,4-BQ, estimated from the adduct levels and reaction-rate constants, were 2- to 4-fold higher in the mouse than in the rat. No adducts of 4,4{prime}-diphenoquionone were detected, in vivo. Background levels of 1,2-BQ and 1,4-BQ adducts were observed with proteins from humans, rats and mice, stemming from the dietary and endogenous sources of BQ precursors.« less
  • Reactive oxygen species (ROS) have been recognized as key molecules, which can selectively modify proteins and therefore regulate cellular signalling including apoptosis. Plumbagin, a naphthoquinone exhibiting antitumor activity, is known to generate ROS and has been found to inhibit the activity of topoisomerase II (Topo II) through the stabilization of the Topo II-DNA cleavable complex. The objective of this research was to clarify the role of ROS and Topo II inhibition in the induction of apoptosis mediated by plumbagin. As determined by the comet assay, plumbagin induced DNA cleavage in HL-60 cells, whereas in a cell line with reduced Topomore » II activity-HL-60/MX2, the level of DNA damage was significantly decreased. The onset of DNA strand break formation in HL-60 cells was delayed in comparison with the generation of intracellular ROS. In HL-60/MX2 cells, ROS were generated at a similar rate, whereas a significant reduction in the level of DNA damage was detected. The pretreatment of cells with N-acetylcysteine (NAC) attenuated plumbagin-induced DNA damage, pointing out to the involvement of ROS generation in cleavable complex formation. These results suggest that plumbagin-induced ROS does not directly damage DNA but requires the involvement of Topo II. Furthermore, experiments carried out using light spectroscopy indicated no direct interactions between plumbagin and DNA. The induction of apoptosis was significantly delayed in HL-60/MX2 cells indicating the involvement of Topo II inhibition in plumbagin-mediated apoptosis. Thus, these findings strongly suggest ROS-mediated inhibition of Topo II as an important mechanism contributing to the apoptosis-inducing properties of plumbagin.« less