1,4-Benzoquinone is a topoisomerase II poison

Benzene is a human carcinogen that induces hematopoietic malignancies. It is believed that benzene does not initiate leukemias directly, but rather generates DNA damage through a series of phenolic metabolites, especially 1,4-benzoquinone. The cellular consequences of 1,4-benzoquinone are consistent with those of topoisomerase II-targeted drugs. Therefore, it has been proposed that the compound initiates specific leukemias by acting as a topoisomerase II poison. This hypothesis, however, has not been supported by in vitro studies. While 1,4-benzoquinone has been shown to inhibit topoisomerase II catalysis, increases in enzyme-mediated DNA cleavage have not been reported. Because of the potential involvement of topoisomerase II in benzene-induced leukemias, we re-examined the effects of the compound on DNA cleavage mediated by human topoisomerase IIalpha. In contrast to previous reports, we found that 1,4-benzoquinone was a strong topoisomerase II poison and was more potent in vitro than the anticancer drug etoposide. DNA cleavage enhancement probably was unseen in previous studies due to the presence of reducing agents in reaction buffers and the incubation of 1,4-benzoquinone with the enzyme prior to the addition of DNA. 1,4-Benzoquinone increased topoisomerase II-mediated DNA cleavage primarily by enhancing the forward rate of scission. In vitro, the compound induced cleavage at DNA sites proximal to a defined leukemic chromosomal breakpoint and displayed a sequence specificity that differed from that of etoposide. Finally, 1,4-benzoquinone stimulated DNA cleavage by topoisomerase IIalpha in cultured human cells. The present findings are consistent with the hypothesis that topoisomerase IIalpha plays a role in the initiation of specific leukemias induced by benzene and its metabolites.     

Characterization of the genotoxicity of anthraquinones in mammalian cells.

Naturally occurring 1,8-dihydroxyanthraquinones are under consideration as possible carcinogens. Here we wanted to elucidate a possible mechanism of their genotoxicity. All three tested anthraquinones, emodin, aloe-emodin, and danthron, showed capabilities to inhibit the non-covalent binding of bisbenzimide Hoechst 33342 to isolated DNA and in mouse lymphoma L5178Y cells comparable to the topoisomerase II inhibitor and intercalator m-amsacrine. In a cell-free decatenation assay, emodin exerted a stronger, danthron a similar and aloe-emodin a weaker inhibition of topoisomerase II activity than m-amsacrine. Analysis of the chromosomal extent of DNA damage induced by these anthraquinones was performed in mouse lymphoma L5178Y cells. Anthraquinone-induced mutant cell clones showed similar chromosomal lesions when compared to the topoisomerase II inhibitors etoposide and m-amsacrine, but were different from mutants induced by the DNA alkylator ethyl methanesulfonate. These data support the idea that inhibition of the catalytic activity of topoisomerase II contributes to anthraquinone-induced genotoxicity and mutagenicity.     

Polychlorinated biphenyl quinone metabolites poison human topoisomerase IIalpha: altering enzyme function by blocking the N-terminal protein gate

Polychlorinated biphenyls (PCBs) are associated with a broad spectrum of human health problems and cause cancer in rodents. In addition, these compounds cause chromosomal aberrations in humans and treated human cells. Although the underlying basis for the chromosomal damage induced by PCBs is not understood, it is believed that these compounds act through a series of phenolic and quinone-based metabolites. Recent studies indicate that several quinones that promote chromosomal damage also act as topoisomerase II poisons. Therefore, the effects of PCB quinone metabolites (including mono and dichlorinated compounds and p- and o-quinones) on the activity of human topoisomerase IIalpha were examined. Results indicate that these compounds are potent topoisomerase IIalpha poisons in vitro and act by adducting the enzyme. They also increase DNA cleavage by topoisomerase IIalpha in cultured human cells. In contrast, incubation of topoisomerase IIalpha with PCB metabolites in the absence of DNA leads to a rapid loss of enzyme activity. On the basis of (1) the differential ability of quinone-treated enzyme to bind circular and linear DNA molecules and (2) the generation of salt-stable noncovalent complexes between topoisomerase IIalpha and circular plasmids in the presence of PCB quinones, it appears that these compounds alter enzyme function (at least in part) by blocking the N-terminal gate of the protein. Finally, exposure to quinones generates a protein species with a molecular mass approximately twice that of a monomeric topoisomerase IIalpha protomer. This finding suggests that PCB quinones block the N-terminal gate by cross-linking the protomer subunits of topoisomerase IIalpha.     

Topoisomerase II and the etiology of chromosomal translocations

Acute leukemias with balanced chromosomal translocations, protean morphologic and immunophenotypic presentations but generally shorter latency and absence of myelodysplasia are recognized as a complication of anti-cancer drugs that behave as topoisomerase II poisons. Translocations affecting the breakpoint cluster region of the MLL gene at chromosome band 11q23 are the most common molecular genetic aberrations in leukemias associated with the topoisomerase II poisons. These agents perturb the cleavage-religation equilibrium of topoisomerase II and increase cleavage complexes. One model suggests that this damages the DNA directly and leads to chromosomal breakage, which may result in untoward DNA recombination in the form of translocations. This review will summarize the evidence for topoisomerase II involvement in the genesis of translocations and extension of the model to acute leukemia in infants characterized by similar MLL translocations.     

Etoposide quinone is a redox-dependent topoisomerase II poison

Etoposide is a topoisomerase II poison that is used to treat a variety of human cancers. Unfortunately, 2-3% of patients treated with etoposide develop treatment-related leukemias characterized by 11q23 chromosomal rearrangements. The molecular basis for etoposide-induced leukemogenesis is not understood but is associated with enzyme-mediated DNA cleavage. Etoposide is metabolized by CYP3A4 to etoposide catechol, which can be further oxidized to etoposide quinone. A CYP3A4 variant is associated with a lower risk of etoposide-related leukemias, suggesting that etoposide metabolites may be involved in leukemogenesis. Although etoposide acts at the enzyme-DNA interface, several quinones poison topoisomerase II via redox-dependent protein adduction. The effects of etoposide quinone on topoisomerase IIα-mediated DNA cleavage have been examined previously. Although findings suggest that the activity of the quinone is slightly greater than that of etoposide, these studies were carried out in the presence of significant levels of reducing agents (which should reduce etoposide quinone to the catechol). Therefore, we examined the ability of etoposide quinone to poison human topoisomerase IIα in the absence of reducing agents. Under these conditions, etoposide quinone was ～5-fold more active than etoposide at inducing enzyme-mediated DNA cleavage. Consistent with other redox-dependent poisons, etoposide quinone inactivated topoisomerase IIα when incubated with the protein prior to DNA and lost activity in the presence of dithiothreitol. Unlike etoposide, the quinone metabolite did not require ATP for maximal activity and induced a high ratio of double-stranded DNA breaks. Our results support the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis.     

Cytosine arabinoside lesions are position-specific topoisomerase II poisons and stimulate DNA cleavage mediated by the human type II enzymes.

Cytosine arabinoside (araC) is an important drug used for the treatment of human leukemias. In order to exert its cytotoxic effects, araC must be incorporated into chromosomal DNA. Although specific DNA lesions that involve base loss or modification stimulate nucleic acid cleavage mediated by type II topoisomerases, the effects of deoxyribose sugar ring modification on enzyme activity have not been examined. Therefore, the effects of incorporated araC residues on the DNA cleavage/religation equilibrium of human topoisomerase IIalpha and beta were characterized. AraC lesions were position-specific topoisomerase II poisons and stimulated DNA scission mediated by both human type II enzymes. However, the positional specificity of araC residues differed from that previously reported for other cleavage-enhancing DNA lesions. Finally, additive or synergistic increases in DNA cleavage were observed in the presence of araC lesions and etoposide. These findings broaden the range of DNA lesions known to alter topoisomerase II function and raise the possibility that this enzyme may mediate some of the cellular effects of araC.     

The 3,4-oxide is responsible for the DNA binding of benzo[ghi]perylene, a polycyclic aromatic hydrocarbon without a "classic" bay-region

The polycyclic aromatic hydrocarbon (PAH) benzo[ghi]perylene (BghiP) lacks a "classic" bay-region and is therefore unable to form vicinal dihydrodiol epoxides thought to be responsible for the genotoxicity of carcinogenic PAHs like benzo[a]pyrene. The bacterial mutagenicity of BghiP increases considerably after inhibition of the microsomal epoxide hydrolase (mEH) indicating arene oxides as genotoxic metabolites. Two K-region epoxides of BghiP, 3,4-epoxy-3,4-dihydro-BghiP (3,4-oxide) and 3,4,11,12-bisepoxy-3,4,11,12-tetrahydro-BghiP (3,4,11,12-bisoxide) identified in microsomal incubations of BghiP are weak bacterial mutagens in strain TA98 of Salmonella typhimurium with 5.5 and 1.5 his+-revertant colonies/nmol, respectively. After microsomal activation of BghiP in the presence of calf thymus DNA three DNA adducts were detected using 32P-postlabeling. The total DNA binding of 2.1 fmol/microg DNA, representing 7 adducts in 10(7) nucleotides, was raised 3.6-fold when mEH was inhibited indicating arene oxides as DNA binding metabolites. Co-chromatography revealed the identity between the main adduct of metabolically activated BghiP and the main adduct of the 3,4-oxide. DNA adducts of BghiP originating from the 3,4,11,12-bisoxide were not found. Therefore, a K-region epoxide is proposed to be responsible for the genotoxicity of BghiP and possibly of other PAHs without a "classic" bay-region.     

Evidence for activation of carcinogenic o-anisidine by prostaglandin H synthase: 32P-postlabelling analysis of DNA adduct formation.

2-Methoxyaniline (o-anisidine) is a urinary bladder carcinogen in both mice and rats. Since the urinary bladder contains substantial peroxidase activity, we examined the ability of prostaglandin H synthase (PHS), a prominent enzyme in the urinary bladder, to activate this carcinogen to metabolites binding to macromolecules. Using [14C]-labeled o-anisidine, we observed substantial PHS-dependent binding of o-anisidine to protein, DNA and polydeoxyribonucleotides [poly(dX)]. This binding is inhibited by radical scavengers glutathione, ascorbate and NADH. The nuclease P1 and 1-butanol extraction enrichment procedure of the 32P-postlabeling analysis of DNA modified by activated o-anisidine provide evidence that covalent binding to DNA is the principal type of DNA modification. Deoxyguanosine is determined to be the major target for binding of o-anisidine in DNA. The possibility that o-anisidine is carcinogenic to the rodent urinary bladder via its activation by bladder PHS is suggested. The results presented here are the first report demonstrating a PHS-mediated activation of o-anisidine to reactive species forming covalent DNA adducts.     

Binding of ATP to human DNA topoisomerase I resulting in an alteration of the conformation of the enzyme.

It has been known for some time that ATP inhibits the DNA relaxation activity of human DNA topoisomerase I. However, the underlying mechanism of this inhibitory effect remains largely unknown. Using filter binding assays, the binding of human DNA topoisomerase I to DNA was decreased in the presence of ATP. This result suggests that the inhibition of DNA relaxation activity of human DNA topoisomerase I by ATP is at the binding step rather than at the nicking or resealing step. DNA topoisomerase I cleavage assay further supports this notion. ATP-agarose binding and UV cross-linking assays also demonstrate that ATP directly and specifically binds human DNA topoisomerase I. To address whether the ATP binding results in conformational changes in human DNA topoisomerase I, various proteases were employed for detecting potential protein conformational changes. Our results indicated that the proteolytic susceptibilities of trypsin and chymotrypsin were altered in the presence of ATP. The result suggests that the conformation of human DNA topoisomerase I was altered upon ATP binding. In addition, the binding between ATP and human DNA topoisomerase I was also reduced by increasing concentrations of DNA. Our data suggests that human DNA topoisomerase I exhibits at least two incompatible conformations. One conformation is in the form of a topoisomerase I-ATP complex, which inhibits DNA relaxation activity of human DNA topoisomerase I, and the other, a topoisomerase I-DNA complex, which exerts DNA relaxation activity. Our studies identify the role of ATP in the regulation of human DNA topoisomerase I and provide a substantial implication of how human DNA topoisomerase I compromises its versatile functions.     

Interpretation of the biological relevance of genotoxicity test results: the importance of thresholds

Despite recent improvements in genotoxicity protocols, we have observed an increase in the occurrence of positive results, particularly in chromosomal aberration tests in vitro, yet very few of these are accompanied by positive responses in vivo. Thus, the positive results may not be biologically relevant either for rodents or humans in vivo, but how should we determine "biological relevance"? Chemicals that produce thresholded dose-responses may well not pose a genotoxic risk at low (relevant to human) exposures, but thresholds should not just be "seen"; there must be an explanation and understanding of the underlying mechanism. In addition to extremes of pH, ionic strength and osmolality, as have been identified previously, such mechanisms include indirect genotoxicity resulting from interaction with non-DNA targets, chemicals/metabolites which are inherently genotoxic but which, at low concentrations, are effectively conjugated and unable to form adducts, and production of specific metabolites under in vitro conditions that are not formed in rodents or humans in vivo. If such thresholded mechanisms can be identified at exposures which are well in excess of expected human exposure, then there may be a strong argument that the positive results are not biologically relevant.     

DNA adduction by polychlorinated biphenyls: adducts derived from hepatic microsomal activation and from synthetic metabolites

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants and complete carcinogens in rodents. Metabolism of lower chlorinated congeners with rat liver microsomes was investigated in earlier studies and DNA adduction was also reported. The current study was designed to compare DNA adducts formed after bioactivation of PCBs with rat, mouse and human hepatic microsomes, and to investigate the role of quinoid PCB metabolites in DNA adduct formation. Eight congeners ranging from mono- to hexachlorinated biphenyls were tested. Metabolites obtained through microsomal bioactivation as well as synthetic quinoid metabolites of 4-monochlorobiphenyl (4-CB) were incubated with calf-thymus DNA (CT-DNA), and the resulting adducts were analyzed by the 32P-post-labelling method. DNA adducts were formed with mono- di- and tri-chlorinated congeners, but not with higher chlorinated congeners. Similar adduct patterns were observed for 2-monochlorobiphenyl (2-CB) activated with hepatic microsomes from rat, mouse and human, while 4-CB, 3,4-dichlorobiphenyl (3,4-CB) and 3,4,5-trichlorobiphenyl (3,4,5-CB) showed similar patterns for two out of the three microsomal systems tested. 4,4' -trichlorobiphenyl (4,4' -CB) showed different adduct patterns in all microsomal systems. Higher adduct levels were obtained with the rodent microsomes compared with human microsomes and were related to higher cytochrome P450 activity. When adducts derived from microsomal activation of 4-CB were compared by co-chromatography with those derived from the incubation of DNA with synthetic 2-(4' -chlorophenyl)-1,4-benzoquinone (4-BQ), one adduct co-migrated in three different chromatography systems. This study demonstrates that rodents as well as human hepatic enzymes metabolize lower chlorinated biphenyl congeners to reactive intermediates that form DNA adducts in vitro and shows that the para-quinone metabolites of PCBs are, in part, involved in direct DNA adduction.     

1,3-Butadiene working group report

During the Workshop in North Carolina, the in vivo metabolism, adduct formation and genotoxicity data available from rodent and human exposure to 1,3-butadiente (BD) were reviewed and they are summarized in the present report. BD is metabolized by cytochrome P-450-dependent monoxygenases to the primary metabolite 1,2-epoxybutene-3 (epoxybutene, EB). EB is subjected to further metabolism: oxidation to 1,2:3,4-diepoxybutane (DEB), hydrolysis to 3-butene-1,2-diol and conjugation to glutathione. The first pathway seems to prevail in mice while the latter is characteristic for rats and possibly for humans. Species differences exist in adduct formation of the monoepoxide to hemoglobin, for which the following pattern has been found: mice > rats > humans. Genotoxity of BD was found in mice with all applied tests; however, negative results were obtained in rats. In exposed humans, the cytogenetic studies in peripheral blood lymphocytes did not show genotoxic effects, although one report described elevated hprt variant levels in peripheral blood lymphocytes of exposed workers.

It was concluded that the presently available data are insufficient for the application of the parallelogram model to estimate genetic risk for humans. As an alternative approach, a tentative estimate of the doubling dose for induction of hprt mutations in somatic cells of mice and men was performed and the calculated values were surprisingly similar, i.e. 9000 ppmh. However, this estimate is burdened with a number of caveats which were discussed in detail. The working group identified a series of urgent research needs to provide the appropriate data for the application of the parallelogram model, such as identification of metabolic pathways in different rodent species and humans, metabolic studies in mice, rats and humans considering metabolic polymorphisms, studies of adducts to DNA and hemoglobin especially of DEB and other butadiene metabolites in rodents and humans, studies of mutational spectra (mutational fingerprinting) in somatic and germinal cells, confirmation of the human hprt mutation data, confirmation of the rodent malformation data, dose-response studies in rodent germ cell tests and studies on repair kinetics of mono-adducts induced by EB as opposed to repair of cross-links produced by DEB. Finally, it was suggested that the original parallelogram consisting of data from somatic cell studies in rodents and humans plus studies of heritable effects in rodents to extrapolate to germ cell risk for humans should be supplemented with studies in sperm of experimental animals and exposed men.     

In vitro genotoxicity test approaches with better predictivity: summary of an IWGT workshop

Improving current in vitro genotoxicity tests is an ongoing task for genetic toxicologists. Further, the question on how to deal with positive in vitro results that are demonstrated to not predict genotoxicity or carcinogenicity potential in rodents or humans is a challenge. These two aspects were addressed at the 5th International Workshop on Genotoxicity Testing (IWGT) held in Basel, Switzerland, on August 17-19, 2009. The objectives of the working group (WG) were to make recommendations on the use of cell types or lines, if possible, and to provide evaluations of promising new approaches. Results obtained in rodent cell lines with impaired p53 function (L5178Y, V79, CHL and CHO cells) and human p53-competent cells (peripheral blood lymphocytes, TK6 and HepG2 cells) suggest that a reduction in the percentage of non-relevant positive results for carcinogenicity prediction can be achieved by careful selection of cells used without decreasing the sensitivity of the assays. Therefore, the WG suggested using p53- competent - preferably human - cells in in vitro micronucleus or chromosomal aberration tests. The use of the hepatoma cell line HepaRG for genotoxicity testing was considered promising since these cells possess better phase I and II metabolizing potential compared to cell lines commonly used in this area and may overcome the need for the addition of S9. For dermally applied compounds, the WG agreed that in vitro reconstructed skin models, once validated, will be useful to follow up on positive results from standard in vitro assays as they resemble the properties of human skin (barrier function, metabolism). While the reconstructed skin micronucleus assay has been shown to be further advanced, there was also consensus that the Comet assay should be further evaluated due to its independence from cell proliferation and coverage of a wider spectrum of DNA damage.     

Prevalence of genotoxic chemicals among animal and human carcinogens evaluated in the IARC Monograph series.

To determine whether genotoxic and non-genotoxic carcinogens contribute similarly to the cancer burden in humans, an analysis was performed on agents that were evaluated in Supplements 6 and 7 to the IARC Monographs for their carcinogenic effects in humans and animals and for the activity in short-term genotoxicity tests. The prevalence of genotoxic carcinogens on four groups of agents, consisting of established human carcinogens (group 1, n = 30), probable human carcinogens (group 2A, n = 37), possible human carcinogens (group 2B, n = 113) and on agents with limited evidence of carcinogenicity in animals (a subset of group 3, n = 149) was determined. A high prevalence in the order of 80 to 90% of genotoxic carcinogens was found in each of the groups 1, 2A and 2B, which were also shown to be multi-species/multi-tissues carcinogens. The distribution of carcinogenic potency in rodents did not reveal any specific characteristic of the human carcinogens in group 1 that would differentiate them from agents in groups 2A, 2B and 3. The results of this analysis indicate that (a) an agent with unknown carcinogenic potential showing sufficient evidence of activity in in vitro/in vivo genotoxicity assays (involving as endpoints DNA damage and chromosomal/mutational damage) may represent a hazard to humans; and b) an agent showing lack of activity in this spectrum of genotoxicity assays should undergo evaluation for carcinogenicity by rodent bioassay, in view of the present lack of validated short-term tests for non-genotoxic carcinogens. Overall, this analysis implies that genotoxic carcinogens add more to the cancer burden in man than non-genotoxic carcinogens. Thus, identification of such genotoxic carcinogens and subsequent lowering of exposure will remain the main goal for primary cancer prevention in man.     

A review of the genotoxicity of 1,2-dichloroethane (EDC)

1,2-Dichloroethane (EDC, CAS#107-06-2) is a high production volume halogenated aliphatic hydrocarbon that is used mainly in the manufacture of vinyl chloride. EDC has been found in ambient and residential air samples, as well as in groundwater, surface water and drinking water. EDC has been well-studied in a variety of genotoxicity assays, and appears to involve the metabolic activation of the parent compound. We critically evaluated the genotoxicity data of EDC and its metabolites as part of an evaluation of carcinogenic mechanisms of action of EDC. EDC is genotoxic in multiple test systems via multiple routes of exposure. EDC has been shown to induce DNA adduct formation, gene mutations and chromosomal aberrations in the presence of key activation enzymes (including CYP450s and/or GSTs) in laboratory animal and in vitro studies. EDC was negative for clastogenesis as measured by the micronucleus assay in mice. In general, an increased level of DNA damage is observed related to the GSH-dependent bioactivation of EDC. Increased chromosomal aberrations with increased CYP450 expression were suggestive of a role for the oxidative metabolites of EDC in inducing chromosomal damage. Taken together, these studies demonstrate that EDC exposure, in the presence of key enzymes (including CYP450s and/or GSTs), leads to DNA adduct formation, gene mutations and chromosomal aberrations.     

Interaction of benzoquinone- and hydroquinone-derivatives of lower chlorinated biphenyls with DNA and nucleotides in vitro

Commercial polychlorinated biphenyls (PCBs) are complete carcinogens in rodents, however, their initiating (DNA damaging) activity has not been conclusively demonstrated. In the present study, we reacted synthetic 2-phenyl-1,4-benzoquinones (BQ) and 2-phenyl-1,4-hydroquinones (HQ) of 2-chloro-, 3-chloro-, 4-chloro-, 3,4-dichloro-, and 3,4,5-trichlorobiphenyls with calf thymus DNA and individual deoxynucleoside-3'-monophosphates for 4 h at 37 degrees C. Analysis of DNA adducts resulting from BQ and HQ derivatives of the test congeners by 32P-postlabeling showed essentially similar adduct patterns. However, the adduct pattern and reactivity differed with the congener used. Quantitatively, 2-chloro-BQ/HQ produced in the highest DNA adduct levels and 3,4,5-chloro-BQ/HQ was the least reactive. Chromatographic comparison of DNA and nucleotide adducts derived from 4-chloro-BQ revealed that cytosine, adenine, and thymidine in the DNA accounted for most of the DNA adducts. Interestingly, none of the adducts in DNA were guanine-derived, even though this mononucleotide was highly reactive. These results suggest that both BQ and HQ derivatives of PCBs are capable of covalently binding to DNA, and chromatographic similarity in adduct patterns resulting from these two metabolites suggest possible involvement of intermediary semiquinone radicals. Experiments are underway to determine their in vivo significance.     

Estrogen, DNA damage and mutations

Estrogen administration to rodents results in various types of DNA damage and ultimately leads to tumors in estrogen-responsive tissues. Yet these hormones have been classified as nonmutagenic, because they did not induce mutations in classical bacterial and mammalian mutation assays. In this review, we have discussed the induction by estrogens of DNA and chromosomal damage and of gene mutations, because the classical assays were designed to uncover mutations only at one specific locus and could not have detected other types of mutations or changes in other genes. Various types of estrogen-induced DNA damage include: (a) direct covalent binding of estrogen quinone metabolites to DNA; (b) enhancement of endogenous DNA adducts by chronic estrogen exposure of rodents; (c) free radical generation by metabolic redox cycling between quinone and hydroquinone forms of estrogens and free radical damage to DNA such as strand breakage, 8-hydroxylation of purine bases of DNA and lipid hydroperoxide-mediated DNA modification. Two different types of chromosomal damage have also been induced by estrogen in vivo and in cells in culture such as numerical chromosomal changes and also structural chromosomal aberrations. Gene mutations have been induced in several cell types in culture either by the parent estrogen or by reactive estrogen quinone metabolites. Furthermore, in estrogen-induced kidney tumors in hamsters, several mutations have been observed in the DNA polymerase beta gene mRNA. Estradiol also induces microsatellite instability in these kidney tumors and in premalignant kidney exposed to estradiol. Although this work is still ongoing, it can be concluded that estrogens are complete carcinogens capable of tumor initiation by mutation potentially in critical genes. The hormonal effects of estrogens may complete the development of tumors.     

Etoposide metabolites enhance DNA topoisomerase II cleavage near leukemia-associated MLL translocation breakpoints

Chromosomal breakage resulting from stabilization of DNA topoisomerase II covalent complexes by epipodophyllotoxins may play a role in the genesis of leukemia-associated MLL gene translocations. We investigated whether etoposide catechol and quinone metabolites can damage the MLL breakpoint cluster region in a DNA topoisomerase II-dependent manner like the parent drug and the nature of the damage. Cleavage of two DNA substrates containing the normal homologues of five MLL intron 6 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase IIalpha, ATP, and either etoposide, etoposide catechol, or etoposide quinone. Many of the same cleavage sites were induced by etoposide and by its metabolites, but several unique sites were induced by the metabolites. There was a preference for G(-1) among the unique sites, which differs from the parent drug. Cleavage at most sites was greater and more heat-stable in the presence of the metabolites compared to etoposide. The MLL translocation breakpoints contained within the substrates were near strong and/or stable cleavage sites. The metabolites induced more cleavage than etoposide at the same sites within a 40 bp double-stranded oligonucleotide containing two of the translocation breakpoints, confirming the results at a subset of the sites. Cleavage assays using the same oligonucleotide substrate in which guanines at several positions were replaced with N7-deaza guanines indicated that the N7 position of guanine is important in metabolite-induced cleavage, possibly suggesting N7-guanine alkylation by etoposide quinone. Not only etoposide, but also its metabolites, enhance DNA topoisomerase II cleavage near MLL translocation breakpoints in in vitro assays. It is possible that etoposide metabolites may be relevant to translocations.     

Repair of DNA damage induced by the mycotoxin alternariol involves tyrosyl-DNA phosphodiesterase 1

Alternariol (AOH) was reported recently to act as a topoisomerase poison. To underline the relevance of topoisomerase targeting for the genotoxic properties of AOH, we addressed the question whether human tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme vital to the repair of covalent DNA-topoisomerase adducts, affects AOH-mediated genotoxicity. The relevance of TDP1 activity on AOH-induced genotoxicity was investigated by the comet assay in human cells overexpressing GFP chimera of TDP1 or the inactive mutant TDP1^H263A as well as in cells subjected to siRNA-mediated knock-down of endogenous TDP1. Cells overexpressing TDP1 exhibited significantly less DNA damage after treatment with AOH in comparison to cells expressing the inactive mutant TDP1^H263A. In accordance with these results, levels of AOH inducing DNA strand breaks were increased in TDP1-suppressed cells in comparison to cells transfected with control siRNA. The specific topoisomerase poisons camptothecin and etoposide caused comparable effects, underlining that TDP1 plays an important role in the repair of topoisomerase-mediated DNA damage. In summary, the repair enzyme TDP1 was identified as a factor for the modulation of AOH-mediated DNA damage in human cells.     

Dexamethasone ameliorates oxidative DNA damage induced by benzene and LPS in mouse bone marrow

Mice were grouped to receive vehicle, dexamethasone (DEX), lipopolysaccharide (LPS), benzene (BZ, 200 mg/kg) and combinations: LPS + DEX, BZ + DEX, LPS + BZ, LPS + DEX + BZ. The DNA damage in bone marrow cells from BZ group was enhanced 2.8-fold measured by nuclear 8-hydroxy-2 '-deoxyguanosine (8-oxodG) and 1.4-fold measured by Comet score (index of DNA breaks) (p < 0.05). In the BZ + DEX group, 8-oxodG level and the Comet score were lowered to 65% and 76% respectively of that in the BZ group (p < 0.05). The BZ + LPS caused a 3.9-fold increase in 8-oxodG and a 1.6-fold increase in the Comet score (p < 0.05). The LPS + DEX + BZ lowered 8-oxodG level and the Comet score to 50% and 78% of the values in the LPS + BZ group, respectively (p < 0.05). Nitrate/nitrite levels in serum were higher after BZ + LPS treatment than after all other treatments. Both 8-oxodG level and the Comet scores were correlated to the serum nitrate/nitrite level across all the treatments (r = 0.55, p < 0.01 and r = 0.69, p < 0.01, respectively). In bone marrow cells the 8-oxodG correlated with the Comet scores (r = 0.80, p < 0.01). We conclude that DEX administration can reduce the DNA damage from BZ treatment and from the combination of BZ and LPS. The correlation of DNA damage with nitrate/nitrite indicates the possible involvement of reactive nitrogen species (RNS) in the interaction between BZ and the inflammatory reaction stimulated by LPS. The 8-oxodG determination is more sensitive than strand break analysis by the Comet assay in bone marrow in vivo in mice for measuring the BZ-induced DNA damage.